Novel molecules of the card-related protein family and uses thereof

ABSTRACT

Novel CARD-12 polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated CARD-12 proteins, the invention further provides CARD-12, fusion proteins, antigenic peptides and anti-CARD-12 antibodies. The invention also provides CARD-12 nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a CARD-12 gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATION INFORMATION

This application is a Continuation Application of U.S. patentapplication Ser. No. 10/449,315, filed May 30, 2003, which is aDivisional application of U.S. patent application Ser. No. 09/841,739,filed Apr. 24, 2001, which is a Continuation-in-part of U.S. patentapplication Ser. No. 09/697,089, filed Oct. 26, 2000, which claimspriority from U.S. Provisional Application Ser. No. 60/161,822, filedOct. 27, 1999. The entire contents of these applications are hereinincorporated by reference.

The contents of the Sequence Listing are submitted herewith on compactdisc in duplicate. Each duplicate disc has a copy of the file “sequencelisting.txt” which is incorporated herein by this reference. This fileis 76 kilobytes and is a copy of the sequence listing filed in U.S.patent application Ser. No. 09/841,739, filed Apr. 24, 2001. This filewas copied onto compact disc on Jun. 6, 2005.

BACKGROUND OF THE INVENTION

In multicellular organisms, homeostasis is maintained by balancing therate of cell proliferation against the rate of cell death. Cellproliferation is influenced by numerous growth factors and theexpression of proto-oncogenes, which typically encourage progressionthrough the cell cycle. In contrast, numerous events, including theexpression of tumor suppressor genes, can lead to an arrest of cellularproliferation.

In differentiated cells, a particular type of cell death calledapoptosis occurs when an internal suicide program is activated. Thisprogram can be initiated by a variety of external signals as well assignals that are generated within the cell in response to, for example,genetic damage. For many years, the magnitude of apoptotic cell deathwas not appreciated because the dying cells are quickly eliminated byphagocytes, without an inflammatory response.

The mechanisms that mediate apoptosis have been intensively studied.These mechanisms involve the activation of endogenous proteases, loss ofmitochondrial function, and structural changes such as disruption of thecytoskeleton, cell shrinkage, membrane blebbing, and nuclearcondensation due to degradation of DNA. The various signals that triggerapoptosis are thought to bring about these events by converging on acommon cell death pathway that is regulated by the expression of genesthat are highly conserved from worms, such as C. elegans, to humans. Infact, invertebrate model systems have been invaluable tools inidentifying and characterizing the genes that control apoptosis. Throughthe study of invertebrates and more evolved animals, numerous genes thatare associated with cell death have been identified, but the way inwhich their products interact to execute the apoptotic program is poorlyunderstood.

Caspases, a class of proteins central to the apoptotic program, areresponsible for the degradation of cellular proteins that leads to themorphological changes seen in cells undergoing apoptosis. Caspases(cysteinyl aspartate-specific proteinases) are cysteine proteases havingspecificity for aspartate at the substrate cleavage site. Generally,caspases are classified as either initiator caspases or effectorcaspases, both of which are zymogens that are activated by proteolysisthat generates an active species. An effector caspase is activated by aninitiator caspase which cleaves the effector caspase. Initiator caspasesare activated by an autoproteolytic mechanism that is often dependentupon oligomerization directed by association of the caspase with anadapter molecule.

Apoptotic signaling is dependent on protein-protein interactions. Atleast three different protein-protein interaction domains, the deathdomain, the death effector domain and the caspase recruitment domain(CARD), have been identified within proteins involved in apoptosis. Afourth protein-protein interaction domain, the death recruiting domain(DRD) was recently identified in murine FLASH (Imai et al. (1999) Nature398:777-85).

Caspases comprise a multi-gene family having at least 12 distinct familymembers (Nicholson (1999) Cell Death and Differentiation 6:1028). Arelatively small fraction of cellular polypeptides (less than 200) arethought to serve as targets for cleavage by caspases. Because many ofthese caspase targets perform key cellular functions, their proteolysisis thought to account for the cellular and morphological events thatoccur during apoptosis. Members of the caspase gene family can bedivided by phylogenetic analysis into two major subfamilies, based upontheir relatedness to ICE (interleukin-1β converting enzyme; caspase-1)and CED-3. Alternate groupings of caspases can be made based upon theirsubstrate specificities.

Many caspases and proteins that interact with caspases possess a CARDdomain. Hofmann et al. ((1997) TIBS 22:155) and others have postulatedthat certain apoptotic proteins bind to each other via their CARDdomains and that different subtypes of CARD domains may confer bindingspecificity, regulating the activity of various caspases, for example.

Apoptosis in mammalian cells is mediated by large protein families thatshare sequence and structural similarity with the core apoptoticproteins of Caenorhabditis elegans (Metzstein et al. (1998) Trends.Genet. 14:410). The nematode CED-4 protein and its human homolog Apaf-1play central roles in apoptosis by transducing death signals to theactivation of caspases. Both CED-4 and Apaf-1 contain an N-terminal CARDdomain that mediates caspase binding and a centrally locatednucleotide-binding site (NBS) domain. Unlike CED-4, Apaf-1 contains aC-terminal WD-40 domain that mediates protein activation in response tothe release of mitochondrial cytochrome c (Zou et al. (1997) Cell90:405; Li et al. (1997) Cell 91:479; Srinivasula et al. (1998) Mol.Cell. 1:949). Additional CED4/Apaf-1 family members include CARD-4, Nod2and CARD-7 (NAC/DEFCAP) (Bertin et al. (1999) J. Biol. Chem. 274:12955;Bertin et al. (2000) J. Biol. Chem. 275:41082; Inohara et al. (2000) J.Biol. Chem. 275:27823; Chu et al. (2001) J. Biol. Chem. 276:9239; Hlianget al. (2001) J. Biol. Chem. 276:9230). CARD4, Nod2 and CARD7 eachcontain NBS domains and effector CARD domains that mediate binding todownstream CARD-containing signaling partners. Both CARD-4 and Nod2assemble together with the CARD protein RICK and induce the activationof NF-kB. Recent evidence suggests that CARD-7 may play a role analogousto Apaf-1 and directly mediate caspase activation. In addition, eachprotein contains extensive leucine-rich repeats (LRR) that have beenproposed to function as binding sites for upstream regulators. Thestructure of CARD-4, Nod2 and CARD-7 is strikingly similar to plantNBS/LRR proteins that induce gene expression and cell death in responseto pathogen infection (Dixon et al. (2000) Proc. Nat'l. Acad. Sci. USA97:8807). Thus, CARD-4, Nod2 and CARD-7 likely play critical roles instress-activated signaling pathways and may be components of the hostinnate immune response.

SUMMARY OF THE INVENTION

The invention features nucleic acid molecules encoding human CARD-12.CARD-12 has a CARD domain, a nucleotide binding site (NBS) domain, and aleucine rich repeat (LRR) domain. These domains are found in a number ofproteins that transmit signals that activate apoptotic and inflammatorypathways in response to stress and other stimuli. Upon activation,CARD-12, like Apaf-1 (Zou et al. (1997) Cell 90:405-413), likely binds anucleotide, allowing CARD-12 to bind to and activate a CARD-containingprotein via a CARD-CARD interaction, leading to modulation of apoptosis.

CARD-12 nucleic acids and polypeptides, as well as modulators of CARD-12activity or expression, are expected to be useful in the modulation ofstress-related, apoptotic and inflammatory responses, e.g., for thetreatment of apoptotic and inflammatory disorders. In addition, CARD-12nucleic acids and polypeptides are expected to be useful in thediagnosis of apoptotic and inflammatory disorders as well as inscreening assays which can be used to identify compounds which can beused to modulate stress-related, apoptotic and inflammatory responses.

Many cytoplasmic plant proteins involved in response to plant pathogens,generally referred to as “R” proteins, have both an NBS domain and anLRR domain (van der Bizen and Jones (1999) Current Biology 8:226-228). Rproteins are involved in both a rapid defense response (hypersensitiveresponse) and more long-term nonspecific resistance (systemic acquiredresistance). The hypersensitive response involves cell and tissue deaththat is localized to the site of infection. The LRR domains of Rproteins are believed to recognize and bind to pathogen proteins,triggering defensive responses. Many R proteins have an amino terminaleffector domain (e.g., a TIR domain or a leucine zipper domain) that isthought to play a role in downstream signaling of events triggered byinfection and, possibly, other stresses.

The R proteins have some structural similarity to APAF-1, a proteinwhich mediates between Bcl-2, a negative regulator of apoptosis, andcaspases. A domain, designated the NB-ARC domain (“nucleotide-bindingadaptor shared by APAF-1, certain R gene products and CED-4”) contains aseries of motifs and residues that are conserved among R proteins andAPAF-1 (van der Bizen and Jones (1999) Current Biology 8:226-228). Inaddition to the NBS domain, APAF-1 has a CARD domain, functionallyanalogous to the effector domain of R proteins, and a WD-40 domain,functionally analogous to the LRR domain of R proteins.

Similar to CARD-12, CARD-4 and CARD-7 have both an NBS domain and an LRRdomain as well as a CARD domain (detailed information concerning CARD-4and CARD-7 can be found in U.S. application Ser. No. 09/245,281, filedFeb. 5, 1999, U.S. Pat. No. 6,369,196; U.S. application Ser. No.09/207,359, filed Dec. 8, 1998, U.S. Pat. No. 6,469,140; U.S.application Ser. No. 09/099,041, filed Jun. 17, 1998, U.S. Pat. No.6,340,576; application Ser. No. 09/019,942, filed Feb. 6, 1998, U.S.Pat. No. 6,033,855; and U.S. application Ser. No. 09/428,252, filed Oct.27, 1999, all of which are incorporated herein by reference). The CARDdomain, which is present in a number of apoptotic signaling molecules,is an effector domain that thought to be involved in homophilicprotein-protein interactions, e.g., with downstream CARD-containingsignaling molecules. For example, the CARD domain of CARD-4 interactswith the CARD domain of RICK (RIP2, CARDIAK), a serine-threonine kinasethat activates NF-κB signaling pathways.

Other proteins structurally related to CARD-12 include NBS-1, Pyrin-1,PCD-1, PCD-2, and PCD-3.

NBS-1 has an NBS domain and a LRR domain, as well as a pyrin domain.Functionally analogous to the CARD domain of CARD-4, CARD-7, andCARD-12, the pyrin domain is an effector domain thought to be involvedin homophilic protein-protein interactions. Pyrin-1 also contains apyrin domain. Detailed information concerning NBS-1 and Pyrin-1 can befound in U.S. application Ser. No. 09/506,067, filed Feb. 17, 2000,which is incorporated herein by reference.

PCD-1, PCD-2, and PCD-3 each contain both an NBS domain and a leucinezipper domain. A leucine zipper domain, like the CARD domain and thepyrin domain, is an effector domain thought to be involved in homophilicprotein-protein interactions. PCD-2 and PCD-3 also each contains LRRdomains. PCD-1, which is truncated at is carboxy terminus, is alsoexpected to contain an LRR domain. Detailed information concerningPCD-1, PCD-2, and PCD-3 can be found in U.S. application Ser. No.09/563,876, filed May 3, 2000, which is incorporated herein byreference.

In general, an NBS domain includes a kinase 1a domain (P-loop), and akinase 2 domain (Walker B box). An LRR domain usually is composed ofseveral leucine rich repeats.

Without being bound by a particular theory, it is possible that CARD-12participates in the network of interactions that lead to caspaseactivity. Human CARD-12 may play a functional role in caspase activationsimilar to that of Apaf-1 (Zou et al., Cell, 90:405-413, 1997). Forexample, upon activation, CARD-12 might bind a nucleotide, thus allowingCARD-12 to bind and activate a CARD-containing caspase via a CARD-CARDinteraction, leading to apoptotic death of the cell.

Accordingly, CARD-12 molecules are useful as modulating agents inregulating a variety of cellular processes including cell growth andcell death. In one aspect, this invention provides isolated nucleic acidmolecules encoding CARD-12 proteins or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection of CARD-12 encoding nucleicacids.

The invention encompasses methods of diagnosing and treating patientswho are suffering from a disorder associated with an abnormal level orrate (undesirably high or undesirably low) of apoptotic cell death,abnormal activity of stress-related pathways of the endoplasmicreticulum (ER), abnormal activity of the Fas/APO-1 receptor complex,abnormal activity of the TNF receptor complex, or abnormal activity of acaspase by administering a compound that modulates the expression ofCARD-12 (at the DNA, mRNA or protein level, e.g., by altering mRNAsplicing) or by altering the activity of CARD-12. Examples of suchcompounds include small molecules, antisense nucleic acid molecules,ribozymes, and polypeptides.

Certain disorders are associated with an increased number of survivingcells, which are produced and continue to survive or proliferate whenapoptosis is inhibited or occurs at an undesirably low rate. CARD-12 andcompounds that modulate the expression or activity of CARD-12 can beused to treat or diagnose such disorders. These disorders include cancer(particularly follicular lymphomas, chronic myelogenous leukemia,melanoma, colon cancer, lung carcinoma, carcinomas associated withmutations in p53, and hormone-dependent tumors such as breast cancer,prostate cancer, and ovarian cancer). Such compounds can also be used totreat viral infections (such as those caused by herpesviruses,poxviruses, and adenoviruses). Failure to remove autoimmune cells thatarise during development or that develop as a result of somatic mutationduring an immune response can result in autoimmune disease. Thus, anautoimmune disorder can be caused by an undesirably low level ofapoptosis. Accordingly, CARD-12 and modulators of CARD-12 activity orexpression can be used to treat autoimmune disorders (e.g., systemiclupus erythematosis, immune-mediated glomerulonephritis, and arthritis).

Many diseases are associated with an undesirably high rate of apoptosis.CARD-12 and modulators of CARD-12 expression or activity can be used totreat or diagnose such disorders. A wide variety of neurologicaldiseases are characterized by the gradual loss of specific sets ofneurons. Such disorders include Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa,spinal muscular atrophy, Huntington's disease, and various forms ofcerebellar degeneration. The cell loss in these diseases does not inducean inflammatory response, and apoptosis appears to be the mechanism ofcell death. In addition, a number of hematologic diseases are associatedwith a decreased production of blood cells. These disorders includeanemia associated with chronic disease, aplastic anemia, chronicneutropenia, and the myelodysplastic syndromes. Disorders of blood cellproduction, such as myelodysplastic syndrome and some forms of aplasticanemia, are associated with increased apoptotic cell death within thebone marrow. These disorders could result from the activation of genesthat promote apoptosis, acquired deficiencies in stromal cells orhematopoietic survival factors, or the direct effects of toxins andmediators of immune responses. Two common disorders associated with celldeath are myocardial infarctions and stroke. In both disorders, cellswithin the central area of ischemia, which is produced in the event ofacute loss of blood flow, appear to die rapidly as a result of necrosis.However, outside the central ischemic zone, cells die over a moreprotracted time period and morphologically appear to die by apoptosis.Additional diseases associated with an undesirably high rate ofapoptosis include: ischemic and hypoxic brain injury, traumatic andexcitotoxic brain damage, neuronal transplantation, acute bacterialmeningitis, kidney ischemia/reperfusion injury, and liver disease.CARD-12 and modulators of CARD-12 may therefore be useful in treatingand diagnosing these conditions.

Populations of cells are often depleted in the event of viral infection,with perhaps the most dramatic example being the cell depletion causedby the human immunodeficiency virus (HIV). Surprisingly, most T cellsthat die during HIV infections do not appear to be infected with HIV.Although a number of explanations have been proposed, recent evidencesuggests that stimulation of the CD4 receptor results in the enhancedsusceptibility of uninfected T cells to undergo apoptosis.

CARD-12 polypeptides, nucleic acids and modulators of CARD-12 expressionor activity can be used to treat inflammatory disorders and immunesystem disorders. The inflammatory and immune disorders include, but arenot limited to, chronic inflammatory diseases and disorders, such asCrohn's disease, reactive arthritis, including Lyme disease,insulin-dependent diabetes, organ-specific autoimmunity, includingmultiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contactdermatitis, psoriasis, graft rejection, graft versus host disease,sarcoidosis, atopic conditions, such as asthma and allergy, includingallergic rhinitis, gastrointestinal allergies, including food allergies,eosinophilia, conjunctivitis, glomerular nephritis, certain pathogensusceptibilities such as helminthic (e.g., leishmaniasis), certain viralinfections, including HIV, and bacterial infections, includingtuberculosis and lepromatous leprosy.

Ischemia is often accompanied by inflammation that causes cell death.Because CARD-12 is expected to play a role in stress-related response,inflammation and apoptosis, CARD-12 polypeptides, nucleic acids, andmodulators of CARD-12 expression or activity can be used to treat cellsdeath accompanying inflammatory responses triggered by ischemia.

Invasive infection with Gram-negative bacteria and Gram-positivebacteria often results in septic shock. CARD-12 may recognize and bindcomponents of Gram-negative bacteria and Gram-positive bacteria or otherinfectious agents (e.g., intracellular parasites), triggering aninflammatory response. Thus, CARD-12 may play a role in innate immunesystem responses that is similar to that of Toll-like receptor 2 (TLR2),a receptor which has some structural similarity to plant R proteins andIL-1R. TLR2 is a signaling receptor that, in association with CD14, isactivated by LPS in a response that requires LPS-binding protein. Theinteraction of TLR2 with LPS leads to TLR2 oligomerization andrecruitment of IRAK (Yang et al. (1998) Nature 395:284-88; Yang et al(1999) J. Immunol. 163:639-43; and Yoshimura et al. (1999) J. Immunol.163:105). Thus, TLR2 is thought to be a direct mediator of signaling byLPS. TLR2 is also thought to mediate cell activation induced bypeptidoglycan and lipoteichoic acid, the main stimulatory components ofGram-positive bacteria (Schwandner et al. (1999) J. Biol. Chem.274:17406-09).

In addition to the aforementioned disorders, CARD-12 polypeptides,nucleic acids, and modulators of CARD-12 expression or activity can beused to treat septic shock and other disorders associated with an innateimmune response. For example, CARD-12 may bind to a component of anintracellular infectious agent or a component of an infectious agentthat is brought into a cell expressing CARD-12, e.g., a component thatenters a cell through a receptor or is expressed by a viral gene.

In addition to the aforementioned disorders, CARD-12 polypeptides,nucleic acids, and modulators of CARD-12 expression or activity can beused to treat disorders of cell signaling and disorders of tissues inwhich CARD-12 is expressed.

The invention features a nucleic acid molecule which is at least 45% (or55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:3, or a complement thereof.

The invention features a nucleic acid molecule which includes a fragmentof at least 150 (300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650,700, 800, 900, 1000, 1300, 1600, 1900, 2100, 2400, 2700, 3000, or 3100)nucleotides of the nucleotide sequence shown in SEQ ID NO:1, or SEQ IDNO:3, or a complement thereof.

In an embodiment, a CARD-12 nucleic acid molecule has the nucleotidesequence shown in SEQ ID NO:1, or SEQ ID NO:3.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ ID NO:2.The fragment can comprise 15, 25, 50, 75, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 contiguous amino acids of SEQ ID NO:2.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the nucleic acid molecule hybridizes toa nucleic acid molecule consisting of SEQ ID NO:1 or SEQ ID NO:3 understringent conditions.

In general, an allelic variant of a gene will be readily identifiable asmapping to the same chromosomal location as the gene.

The invention also includes a nucleic acid molecule encoding a naturallyoccurring polypeptide, wherein the nucleic acid hybridizes to a nucleicacid molecule consisting of SEQ ID NO:3 under stringent conditions(e.g., hybridization in 6× sodium chloride/sodium citrate (SSC) at about60° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.),and wherein the nucleic acid encodes a polypeptide of 1020-1028 aminoacids in length, preferably 1024 amino acids, having a molecular weightof about 116.1 kD prior to post-translational modifications. Thus, theinvention encompasses a nucleic acid molecule which includes thesequence of the protein coding region of a naturally occurring mRNA (orthe corresponding cDNA sequence) that is expressed in a human cell.

Also within the invention are: an isolated CARD-12 protein having anamino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO:2; anisolated CARD-12 protein having an amino acid sequence that is at leastabout 85%, 95%, or 98% identical to the P-loop domain of SEQ ID NO:2(e.g., about amino acid residues 169 to 179 of SEQ ID NO:2); an isolatedCARD-12 protein having an amino acid sequence that is at least about85%, 95%, or 98% identical to the CARD domain of SEQ ID NO:2 (e.g.,about amino acid residues 1 to 88 of SEQ ID NO:2); an isolated CARD-12protein having an amino acid sequence that is at least about 65%,preferably 75%, 85%, 95%, or 98% identical to the nucleotide bindingsite core domain of SEQ ID NO:2 (e.g., about amino acid residues 161 to323 of SEQ ID NO:2); an isolated CARD-12 protein having an amino acidsequence that is at least about 65%, preferably 75%, 85%, 95%, or 98%identical to one or more of the leucine rich repeats of SEQ ID NO:2(e.g., about amino acids residues 762-789, 819-846, 847-874, and 938-965of SEQ ID NO:2); and an isolated CARD-12 protein having an amino acidsequence that is at least about 85%, 95%, or 98% identical to the NAIPhomology region of SEQ ID NO:2 (e.g., about amino acid residues 150 to1024 of SEQ ID NO:2).

Also within the invention are: an isolated CARD-12 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ IDNO:3; an isolated CARD-12 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 85%, 95%, or 98%identical to the P-loop domain encoding portion of SEQ ID NO:3; anisolated CARD-12 protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence at least about 65% preferably 75%, 85%, or95% identical the CARD domain encoding portion of SEQ ID NO:3; anisolated CARD-12 protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence at least about 65% preferably 75%, 85%, or95% identical the NAIP homology encoding portion of SEQ ID NO:3; anisolated CARD-12 protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence at least about 65% preferably 75%, 85%, or95% identical the nucleotide binding site domain encoding portion of SEQID NO:3; an isolated CARD-12 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the LRR domain encoding portion of SEQ ID NO:3or one or more leucine rich repeat encoding portions of SEQ ID NO:3; andan isolated CARD-12 protein which is encoded by a nucleic acid moleculehaving a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:3.

The CARD-12 nucleic acids, polypeptides, and antibodies of the inventionmay be useful for mapping the location of the CARD-12 gene.

Another embodiment of the invention features CARD-12 nucleic acidmolecules which specifically detect CARD-12 nucleic acid molecules,relative to nucleic acid molecules encoding other members of the CARDsuperfamily and/or members of the NBS/LRR superfamily. For example, inone embodiment, a CARD-12 nucleic acid molecule hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, or a complementthereof. In another embodiment, the CARD-12 nucleic acid molecule is atleast 300 (350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1300,1600, 1900, 2100, 2400, 2700, 3000, or 3100) nucleotides in length andhybridizes under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, ora complement thereof. In another embodiment, an isolated CARD-12 nucleicacid molecule comprises the CARD domain encoding portion of SEQ ID NO:3,or a complement thereof. In yet another embodiment, the inventionprovides an isolated nucleic acid molecule which is antisense to thecoding strand of a CARD-12 nucleic acid.

Another aspect of the invention provides a vector, e.g., a recombinantexpression vector, comprising a CARD-12 nucleic acid molecule of theinvention. In another embodiment the invention provides a host cellcontaining such a vector. The invention also provides a method forproducing CARD-12 protein by culturing, in a suitable medium, a hostcell of the invention containing a recombinant expression vector suchthat a CARD-12 protein is produced.

Another aspect of this invention features isolated or recombinantCARD-12 proteins and polypeptides. Preferred CARD-12 proteins andpolypeptides possess at least one biological activity possessed bynaturally occurring human CARD-12, e.g., (1) the ability to formprotein:protein interactions with proteins in the apoptotic signalingpathway; (2) the ability to form CARD-CARD interactions with proteins inthe apoptotic signaling pathway; (3) the ability to bind a CARD-12ligand; and (4) the ability to bind to an intracellular target. Otheractivities include: (1) modulation of cellular proliferation; (2)modulation of cellular differentiation; (3) modulation of cellulardeath; (4) modulation of ER-specific apoptosis pathways; (5) modulationof the NF-kB pathway; (6) modulation of stress-responsive signalingpathways; and (7) modulation of an innate immune response.

The CARD-12 proteins of the present invention, or biologically activeportions thereof, can be operatively linked to a non-CARD-12 polypeptide(e.g., heterologous amino acid sequences) to form CARD-12 fusionproteins, respectively. The invention further features antibodies thatspecifically bind CARD-12 proteins, such as monoclonal or polyclonalantibodies. In addition, the CARD-12 proteins or biologically activeportions thereof can be incorporated into pharmaceutical compositions,which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of CARD-12 activity or expression in a biological sample bycontacting the biological sample with an agent capable of detecting anindicator of CARD-12 activity such that the presence of CARD-12 activityis detected in the biological sample.

In another aspect, the invention provides a method for modulatingCARD-12 activity comprising contacting a cell with an agent thatmodulates (inhibits or stimulates) CARD-12 activity or expression suchthat CARD-12 activity or expression in the cell is modulated. In oneembodiment, the agent is an antibody that specifically binds to CARD-12protein. In another embodiment, the agent modulates expression ofCARD-12 by modulating transcription of a CARD-12 gene, splicing of aCARD-12 mRNA, or translation of a CARD-12 mRNA. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of the CARD-12 mRNA orthe CARD-12 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant CARD-12protein or nucleic acid expression or activity or related to CARD-12expression or activity by administering an agent which is a CARD-12modulator to the subject. In one embodiment, the CARD-12 modulator is aCARD-12 protein. In another embodiment the CARD-12 modulator is aCARD-12 nucleic acid molecule. In other embodiments, the CARD-12modulator is a peptide, peptidomimetic, or other small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a CARD-12 protein; (ii) mis-regulation of a gene encoding aCARD-12 protein; (iii) aberrant RNA splicing; and (iv) aberrantpost-translational modification of a CARD-12 protein, wherein awild-type form of the gene encodes a protein with a CARD-12 activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a CARD-12 protein.In general, such methods entail measuring a biological activity of aCARD-12 protein in the presence and absence of a test compound andidentifying those compounds which alter the activity of the CARD-12protein.

The invention also features methods for identifying a compound whichmodulates the expression of CARD-12 by measuring the expression ofCARD-12 in the presence and absence of a compound.

The invention also features methods for treating disorders associatedwith inappropriate apoptosis (e.g., Alzheimer's diseases or otherneurological disorders associated with neuronal apoptosis) by modulatingthe expression or activity of CARD-12.

The invention also features methods for identifying a compound thatalters (increases or decreases) the binding of CARD-12 (or a CARD domaincontaining portion thereof) to a CARD domain containing protein (e.g.,CARD-5) or a CARD domain containing portion thereof). The methodincludes measuring the binding of the protein (or polypeptides) to eachother in the presence and absence of a test compound and identifying thetest compound as a compound that alters binding if the binding in thepresence of test compound differs from the binding in the absence of thetest compound.

The invention also features a method for identifying a compound thatbinds to the NBS domain of CARD-12 by measuring the binding of a testcompound to a polypeptide comprising the NBS domain of CARD-12. Thebinding can be measured in the presence of a nucleotide (e.g., an NTPsuch as ATP) for a competitive binding assay. Alternatively, the bindingcan be measured in the absence of a nucleotide that binds to the NBSsite.

The invention also features a method for identifying compounds thatalter (increase or decrease) CARD-12 mediated apoptosis. The methodsinclude measuring apoptosis in the presence and absence of a testcompound in cells expressing CARD-12 and in cells not expressing CARD-12(or expressing less CARD-12). The CARD-12 expressed by the cell can beencoded by a vector introduced into the cell. Thus, the cells canover-express CARD-12. A compound that alters apoptosis in the cellsexpressing CARD-12, but not in the cells not expressing CARD-12 (orexpressing less CARD-12), the compound is a candidate CARD-12 specificmodulator of apoptosis.

Compounds that increase the binding of CARD-12 to CARD-5 can be used tosupplement chemotherapeutic agents.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E depict the cDNA sequence (SEQ ID NO:1) and predicted aminoacid sequence (SEQ ID NO:2) of human CARD-12. The open reading frame ofCARD-12 (SEQ ID NO:1) extends from nucleotide 36 to nucleotide 3107 ofSEQ ID NO:1 (SEQ ID NO:3).

FIGS. 2A-2E depict a predicted nucleotide sequence (SEQ ID NO:4) andpredicted amino acid sequence (SEQ ID NO:5) of human CARD-12, based uponthe CARD-12 sequence derived from a genomic clone. The complimentary DNAis also depicted (SEQ ID NO:12).

FIG. 3 depicts a hydropathy plot of CARD-12. Relatively hydrophobicresidues are above the dashed horizontal line, and relativelyhydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and N-glycosylation (Ngly) site are indicated by shortvertical lines just below the hydropathy trace.

FIG. 4 depicts a plot showing the predicted structural features ofCARD-12. This figure shows the predicted alpha regions (Garnier-Robsonand Chou-Fasman), the predicted beta regions (Garnier-Robson andChou-Fasman), the predicted turn regions (Garnier-Robson andChou-Fasman) and the predicted coil regions (Garnier-Robson). Alsoincluded in the figure is a hydrophilicity plot (Kyte-Doolittle), thepredicted alpha and beta-amphipathic regions (Eisenberg), the predictedflexible regions (Karplus-Schulz), the predicted antigenic index(Jameson-Wolf) and the predicted surface probability plot (Emini).

FIG. 5A depicts an alignment of amino acids 2-88 of human CARD-12 (aminoacid residues 2-88 of SEQ ID NO:2) with a CARD domain (SEQ ID NO:7)derived from a hidden Markov model.

FIG. 5B depicts an alignment of amino acids 764-791 of human CARD-12(amino acid residues 764-791 of SEQ ID NO:2) with a consensus leucinerich repeat (SEQ ID NO:8) derived from a hidden Markov model.

FIG. 5C depicts an alignment of amino acids 821-848 of human CARD-12(amino acid residues 821-848 of SEQ ID NO:2) with a consensus leucinerich repeat (SEQ ID NO:8) derived from a hidden Markov model.

FIG. 5D depicts an alignment of amino acids 849-872 of human CARD-12(amino acid residues 849-872 of SEQ ID NO:2) with a consensus leucinerich repeat (SEQ ID NO:8) derived from a hidden Markov model.

FIG. 5E depicts an alignment of amino acids 938-965 of human CARD-12(amino acid residues 938-965 of SEQ ID NO:2) with a consensus leucinerich repeat (SEQ ID NO:8) derived from a hidden Markov model.

FIGS. 6A-6C depict an alignment of amino acids 150-1024 of human CARD-12(amino acid residues 150-1024 of SEQ ID NO:2) with amino acids 451-1232of neuronal AIP (SEQ ID NO:9). The consensus sequence and majoritysequence are also depicted in (SEQ ID NOs:10 and 11, respectively).

FIG. 7 depicts an alignment of the CARD domain of human CARD-12 (aminoacids 1-88 of SEQ ID NO:2; SEQ ID NO:13) with the CARD domains of CARD-4(SEQ ID NO:14), CARD-7 (SEQ ID NO:15), and Apaf-1 (SEQ ID NO:16).

FIG. 8 depicts schematic drawings of the domain structures of humanCARD-12, human CARD-4, human CARD-7, human Nod2, and Apaf-1.

FIG. 9 depicts the results of a mammalian two-hybrid assay used toidentify CARD domains that interact with the CARD domain of CARD-12.

FIGS. 10A-10C depict the results of co-immunopreciptation analysis. FIG.10A: After 24 hrs, extracts were prepared and immunoprecipitated (IP)with a monoclonal antibody to the T7 epitope. The immunoprecipitateswere analyzed by SDS-PAGE and immunoblotted with an anti-myc polyclonalantibody. FIG. 10B: The cellular extracts were also immunoblotted (WB)with anti-T7 antibody. FIG. 10C: Extracts were also immunoprecipitatedwith a monoclonal antibody to myc, followed by WB with an anti-mycpolyclonal antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the identification of a cDNAsequence encoding a human CARD-12 protein. A nucleotide sequenceencoding a human CARD-12 protein is shown in FIGS. 1A-1E (SEQ ID NO:1;SEQ ID NO:3 includes the open reading frame only). A predicted aminoacid sequence of CARD-12 protein is also shown in FIGS. 1A-1E (SEQ IDNO:2).

EXAMPLE 1 Identification and Characterization of CARD-12

A BAC clone (GenBank™ Accession Number AL121653) was searched toidentify potential exons. The predicted protein sequences were thensearched using a profile of known CARD domains. This search led to theidentification of a sequence predicted to encode a CARDdomain-containing protein later identified as CARD-12. FIGS. 2A-2Edepict a nucleotide sequence (SEQ ID NO:4) assembled from the BAC clonewhich includes a predicted open reading frame (SEQ ID NO:6; nucleotides1-3612 of SEQ ID NO:4) encoding a 1204 amino acid protein (SEQ ID NO:5).

The CARD-12 sequence assembled from the BAC clone was used to furthercharacterize the CARD-12 cDNA sequence. A search of a proprietaryelectronic cDNA database (created from a human lymphocyte library)using-sequences derived from the 3′ end of SEQ ID NO:4 led to theidentification of a cDNA containing the 3′ end of the CARD-12 cDNA. Thesequence of the 5′ end of the CARD-12 cDNA was determined by a search ofthe Incyte (Palo Alto, Calif.) Life Gold Templates cDNA electronicdatabase using sequences from the 5′ end of SEQ ID NO:4. A cDNA sequencewas identified (Incyte clone number 328193) that contained 366nucleotides of the 5′ portion of the CARD-12 cDNA. Primers were designedcorresponding to the 5′ untranslated region and the 3′ carboxy terminuscoding region of CARD-12. PCR amplification using these primers wasperformed on a placenta cDNA library. The sequencing of PCR products ledto the identification of the cDNA sequence of CARD-12 (SEQ ID NO:1).

The CARD-12 cDNA sequence (SEQ ID NO:1) has some differences compared tothat predicted by the assembly of the predicted CARD-12 exons of the BACclone (SEQ ID NO:4). These differences are generally located at the 5′and 3′ end of the cDNA. Where the CARD-12 sequences depicted in SEQ IDNO:1 and SEQ ID NO:4 differ, SEQ ID NO:1 corresponds to the CARD-12 cDNAsequence.

FIGS. 1A-1E depict the sequence of a 3133 nucleotide cDNA (SEQ ID NO:1)which includes a predicted open reading frame (SEQ ID NO:3; nucleotides36-3107 of SEQ ID NO:1) encoding a 1024 amino acid human CARD-12 protein(SEQ ID NO:2). Human CARD-12 is predicted to be an intracellularprotein.

The predicted amino acid sequence of human CARD-12 was compared to aminoacid sequences of known proteins and various motifs were identified. The1024 amino acid human CARD-12 protein includes two N-glycosylation sites(e.g., about amino acid residues 539-542 and 764-767 of SEQ ID NO:2); aglycosaminoglycan attachment site (e.g., about amino acid residues171-174 of SEQ ID NO:2); four cAMP-and cGMP-dependent protein kinasephosphorylation site (e.g., about amino acid residues 60-63, 228-231,453-456, and 985-988 of SEQ ID NO:2); 11 protein kinase Cphosphorylation sites (e.g., about amino acid residues 72-74, 171-173,188-190, 226-228, 403-405, 536-538, 566-568, 665-667, 689-691, 710-712,and 973-975 of SEQ ID NO:2); 20 casein kinase II phosphorylation sites(e.g., about amino acid residues 72-75, 94-97, 133-136, 215-218,279-282, 365-368, 415-418, 445-448, 460-463, 479-482, 497-500, 541-544,553-556, 607-610, 665-668, 725-728, 742-745, 851-854, 920-923, and973-976 of SEQ ID NO:2); a tyrosine kinase phosphorylation site (e.g.,about amino acid residues 71-78 of SEQ ID NO:2); 15 N-myristoylationsites (e.g., about amino acid residues 62-67, 156-161, 187-192, 211-216,291-296, 380-385,516-521, 618-623, 699-704, 754-759, 760-765, 894-899,928-933, 946-951, and 959-964 of SEQ ID NO:2); an amidation site (e.g.,about amino acid residues 451-454 of SEQ ID NO:2); and anATP/GTP-binding site motif A (P-loop) (e.g., about amino acid residues169-179 of SEQ ID NO:2).

FIG. 3 depicts a hydropathy plot of CARD-12. Relatively hydrophobicresidues are above the dashed horizontal line, and relativelyhydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and N-glycosylation (Ngly) site are indicated by shortvertical lines just below the hydropathy trace.

A plot showing the predicted structural features of CARD-12 is presentedin FIG. 4. This figure shows the predicted alpha regions (Garnier-Robsonand Chou-Fasman), the predicted beta regions (Garnier-Robson andChou-Fasman), the predicted turn regions (Garnier-Robson andChou-Fasman) and the predicted coil regions (Garnier-Robson). Alsoincluded in the figure is a hydrophilicity plot (Kyte-Doolittle), thepredicted alpha and beta-amphipathic regions (Eisenberg), the predictedflexible regions (Karplus-Schulz), the predicted antigenic index(Jameson-Wolf) and the predicted surface probability plot (Emini).

An analysis of the predicted CARD-12 amino acid sequence showed it tocontain a CARD domain (e.g., about amino acid residues 1-88 of SEQ IDNO:2), a nucleotide binding site (NBS) domain (e.g., about amino acidresidues 161-323 of SEQ ID NO:2), and three leucine rich repeats (LRR;e.g., about amino acid residues 762-789, 819-846, 847-874, and 938-965of SEQ ID NO:2) which form a LRR domain (e.g., about amino acid residues762-965 of SEQ ID NO:2). Within the predicted NBS domain there is akinase 1a domain (P-loop) (e.g., about amino acid residues 169-179 ofSEQ ID NO:2) and a kinase 2 domains (e.g., about amino acid residues245-248 of SEQ ID NO:2).

FIG. 5A depicts an alignment of amino acids 2-88 of human CARD-12 (aminoacid residues 2-88 of SEQ ID NO:2) with a consensus CARD domain (SEQ IDNO:7) derived from a HMM.

FIGS. 5B-5E each depict an alignment of one of the four leucine richrepeats within the LRR domain of CARD-12 (amino acid residues 764-791 ofSEQ ID NO:2 (FIG. 5B), amino acid residues 821-848 of SEQ ID NO:2 (FIG.5C), amino acid residues 849-872 of SEQ ID NO:2 (FIG. 5D), and aminoacid residues 938-965 of SEQ ID NO:2 (FIG. 5E)) with a consensus LRR(SEQ ID NO:8) derived from a HMM.

The domain alignments depicted in FIGS. 5A-5E were identified byhomology searching using consensus domains derived from hidden Markovmodels (HMMs). HMMs can be used to perform multiple sequence alignmentand very sensitive database searching, using statistical descriptions ofa domain's consensus sequence. For more information on HMM searches,see, e.g., the Pfam website maintained in several locations, e.g. byWashington University in St. Louis Mo. In the alignments of FIGS. 5A-5Ea single letter amino acid designation at a position on the line betweenthe CARD-12 sequence and the HMM-generated consensus domain sequenceindicates an exact match between the two. A “+” in this middle lineindicates a conservative substitution at the particular residue ofCARD-12. Amino acid residues located in the domains identified by theHMM search may be important for the appropriate functioning of theCARD-12 protein. For this reason, amino acid substitutions with respectto the sequence of SEQ ID NO:2 that are outside of the domainshomologous to HMM consensus domains may be less detrimental to theactivity of the CARD-12 protein.

The C-terminal portion of CARD-12 (amino acids 150 to 1024 of SEQ IDNO:2) bears some similarity to the N-terminus of neuronal AIP (GenBank™Accession Number Q13075; Roy et al. (1995) Cell 80:167-178). FIGS. 6A-6Cdepict an alignment of amino acids 451-1232 of neuronal AIP (SEQ IDNO:9) and amino acids 150-1024 of human CARD-12.

EXAMPLE 2 Additional Characterization of CARD-12 Domains

CARD-12 includes seven NACHT (NAIP, CIIA, HET-E and TP1) NTPase domains.The seven NACHT NTPase domains are at amino acids 169-186 of SEQ ID NO:2(P-Loop/Walker A Box/Motif I); amino acids 196-220 of SEQ ID NO:2(Walker B Box/Mg⁺⁺ binding domain Motif II); amino acids 229-253 of SEQID NO:2 (Motif III); amino acids 261-282 of SEQ ID NO:2 (Motif IV);amino acids 330-351 of SEQ ID NO:2 (Motif V); amino acids 414-430 of SEQID NO:2 (Motif VI) and amino acids 438-457 of SEQ ID NO:2 (Motif VII).Other members of the NACHT NTPase family include: CARD-4, CARD-7, andNAIP (Koonin et al. (2001) Trends Biochem. Sci. 25:223).

Additional analysis of the leucine-rich repeat domain of CARD-12revealed that the domain extends from amino acid 656-1021 of SEQ ID NO:2and contains 13 leucine-rich repeats (amino acids 656-686, 687-708,711-737, 738-761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930,936-962, 965-993, and 994-1021 of SEQ ID NO:2.

Additional analysis of CARD-12 revealed that the nucleotide binding sitedomain extends from amino acid 169 to amino acid 456 of SEQ ID NO:2.

As discussed above, the CARD domain of CARD-12 extends from amino acid1-88 of SEQ ID NO:2. FIG. 7 depicts an alignment of the CARD domain ofhuman CARD-12 (amino acids 1-88 of SEQ ID NO:2) with the CARD domains ofCARD-4, CARD-7, and Apaf-1.

As discussed above, CARD-12 includes domains present in members of theCED/Apaf-1 family. FIG. 8 depicts schematic drawings of the domainstructures of human CARD-12 as well as human CARD-4, CARD-7, Nod2, andApaf-1, all of which are members of the CED/Apaf-1 family.

Molecules that binding to and alter the activity of the NBS domain ofCARD-12 may be useful for modulating the activity of CARD-12. Forexample, molecules can be tested for their ability to modulate, e.g.,antagonize, the hydrolysis of an NTP, e.g., ATP, by thenucleotide-binding site of CARD-12. Methods of detecting the hydrolysisof ATP by a nucleotide-binding site are described in, for example,Gadsby et al. (1999) Physiol. Rev. 79:S77-S107. Additional assays thatmight be used are described in Li et al., (1996) J. Biol. Chem. 271:28463-28468.

EXAMPLE 3 Expression Analysis

Northern blot analysis of CARD-12 expression using a human adult tissueblot (Clontech, La Jolla, Calif.) revealed that a 3.3-kilobase CARD-12transcript is present in

-   -   human lymphoid tissues, including spleen, peripheral blood        lymphocytes, bone morrow and fetal liver.

An affinity purified polyclonal CARD-12 antibody was used to investigateexpression in primary normal human epithelial cells (Epipanel;Clonetics, Inc.). Affinity-purified CARD-12 antibody was raised inrabbits injected with the 15-mer peptide LWRQESLQSVKNTTE correspondingto residues 527-542 of CARD-12, SEQ ID NO:2 (Research Genetics). Theanalysis revealed that a about 120 kD CARD-12 protein is expressed inhuman renal cortical primary epithelial cells, human mammary primaryepithelial cells, human renal proximal tubule primary epithelial cells,human bronchial primary epithelial cells, and human prostate primaryepithelial cells. Expression was also detected in human primaryepithelial cells, and muscle primary cells.

EXAMPLE 4 CARD-12 Mediates Apoptosis

To determine a possible role for CARD-12 in apoptosis signaling arecombinant adenovirus expressing full length CARD-12 was constructed.Briefly, CARD-12 was expressed using a dual-adenovirus based,tetracycline-regulatable expression system. A similar system haspreviously been shown to work extremely efficiently both in vitro and invivo. CARD-12 was cloned into the adenovirus transfer vector pLE11f,placing the CARD-12 gene under the transcriptional control of thetetracycline-regulatable promoter. An internal downstream ribosome entrysite allowed a modified green fluorescent protein (KGFP) to be expressedoff the same transcript. E1/E3-deleted adenovirus was then generated byhomologous recombination in 911 cells (Fallaux et al. (1996) Hum. GeneTher. 7:215), plaque-purified and protein expression verified by Westernblot. VERO cells were transfected at an MOI of about 20. As a control,cells were similarly transfected with an adenovirus vector expressingKGFP only. VERO cells were plated in 96 well dishes and transfected thefollowing day with adenovirus (MOI of about 20). Cells were fixed (4%paraformaldehyde in 0.15M PBS) 36 hours after transfection. The nucleiwere then stained with Hoescht 3342 and the percentage of apoptoticversus healthy nuclei in transfected cells was scored. Within 36 hoursof transfection with CARD-12 expressing adenovirus, the transfectedcells were undergoing apoptosis, as indicated by rounding up andmembrane blebbing. Additionally, 45.2±4.0% (mean±SE) ofCARD-12-transfected cells had condensed, pyknotic nuclei, whereas only3.8±1.1% of KGFP-transfected cells showed signs of apoptosis.Adenovirus-mediated transfection of either CARD-12 or KGFP in VERO cellsresulted in at least 90% of cells transfected. Thus, CARD-12 is thenovel member of the Apaf-1/CED4 protein family (along with CARD-4,CARD-7, Nod2 and Apaf-1) shown to activate downstream cell deathsignals. As a member of the Apaf-1/CED4 family, transduction of aproapoptotic signal through CARD-12 is likely to be mediated byCARD/CARD interaction with one or more CARD-containing signalingmolecules.

EXAMPLE 5 CARD-12 Interacts with CARD-5 (ASC)

A mammalian two-hybrid analysis was used to identify potential bindingpartners of CARD-12. In this analysis the binding of the N-terminal CARDof CARD-12 to the CARD domains of 23 known proteins was assessed. Theanalysis involved the used of a plasmid, pCMV-CARD-12-CARD/AD,constructed by inserting the CARD domain of CARD-12 (residues 1-83) intopCMV-AD (Stratagene; La Jolla, Calif.). In the assay, 293T cells in6-well plates (35-mm wells) were transfected with the followingplasmids: 750 ng of pCMV-CARD-12/AD, 750 ng of pCMV-BD fused toindividual CARD domains, 250 ng of pFR-Luc firefly reporter(Stratagene), and 250 ng of pRL-TK renilla reporter (Promega). Cellswere harvested 24 hrs after transfection, and firefly luciferaseactivity was determined using the DUAL-LUCIFERASE® Reporter Assay System(Promega). In addition, renilla luciferase activity was determined andused to normalize transfection efficiencies. The results of thetwo-hybrid analysis are shown in FIG. 9. The CARD of CARD-12 interactedwith the CARD of CARD-5, resulting in an 10-fold increase in relativeluciferase activity. The CARD domain of CARD-12 also had a strongself-association, resulting in a 30-fold increase in relative luciferaseactivity (FIG. 9). Co-expression of CARD-12 CARD with other CARD domainsfailed to activate luciferase expression indicating that the CARD ofCARD-12 interacts selectively with the CARD of CARD-5, a protein thatmediates apoptosis induced by chemotherapeutic agents (Masumoto et al.(1999) J. Biol. Chem. 274:33835).

EXAMPLE 6 The Pyrin Domain of CARD-5 Mediates Apoptosis

CARD-5, which consists of an N-terminal PYRIN domain and a C-terminalCARD domain is a proapoptotic protein and is subject tomethylation-induced silencing in a number of breast cancers. This,latter observation suggests that CARD-5 may play a fundamental role incell death. Given that CARD-12 and CARD-5 interact via their respectiveCARDs, the PYRIN domain of CARD-5 may function as its proapoptoticeffector domain. To examine this possibility, adenovirus vectorsexpressing CARD-5 truncation mutants were created. Briefly, CARD-5truncation mutants containing the PYRIN domain or the CARD domain, werecloned into the adenovirus transfer vector pLE11f, placing the gene ofinterest under the transcriptional control of thetetracycline-regulatable promoter. An internal ribosome entry sitedownstream to the gene of interest allows a modified KGFP to beexpressed off the same transcript. E1/E3-deleted adenovirus was thengenerated by homologous recombination in 911 cells, plaque-purified andprotein expression verified by Western blot. VERO cells were transfected(MOI=20) with recombinant adenovirus expressing full length CARD-5(AdTRE-CARD51-195), or either of the PYRIN domain(AdTRE-CARD-5-PYR1-150) or CARD (AdTRE-CARD-5-CARD74-195) of CARD-5alone. Thirty-six hours after transfection cells were fixed and stainedwith the nuclear dye Hoescht 33342 and the percentage of apoptoticversus healthy nuclei in transfected cells was then scored. Western blotfor the FLAG epitope-tag indicate relative levels of expression fromeach vector. Thirty-six hours after transfection of VERO cells with anadenovirus expressing full length CARD-5 60.4+1.6% of cells wereundergoing apoptosis. Interestingly, transfection with the PYRIN domainof CARD-5 alone resulted 66.1±5.4% cell death. Expression of the CARD ofCARD-5 alone resulted in virtually no cell death (2.6±0.6%). Theseresults suggest that a PYRIN domain can play a functional role inapoptosis signaling, and substantiates the emerging hypothesis thatPYRIN-containing proteins represent another important family of proteinsinvolved in transducing the complex signals of apoptosis.

EXAMPLE 7 CARD-12 Interacts with Caspase-1

An additional mechanism by which CARD-12 may cause cell death is by theactivation of upstream caspases via a CARD/CARD interaction. The proformof caspase-1 and caspase-9 both contain an N-terminal CARD. Therefore,caspase-1 and caspase-9 were investigated as possible CARD-12 signalingpartners following transient overexpression in cells. Briefly, 293Tcells transfected with plasmids were lysed in 50 mM Tris, pH 8.0, 120 mMNaCl, 1 mM EDTA, 0.5% Nonidet P-40 buffer and incubated with either a T7(Sigma) or myc monoclonal antibody (SantaCruz Biotechnology, Inc.). Theimmune complexes were precipitated with protein G-Sepharose (AmershamPharmacia Bio), washed extensively, and then subjected toSDS-polyacrylamide gel electrophoresis and immunoblotted with polyclonalantibody to myc (SantaCruz). The results of the co-immunoprecipitationanalysis are shown in FIGS. 10A-10C. Immunoprecipitation of T7-taggedcaspase-1, but not T7-tagged procaspase-9, co-precipitated myc-taggedCARD-12 (FIG. 10A, lanes 1 and 3, respectively). These results indicatea possible role for CARD-12 in caspase-1 signaling. The structuralsimilarity of CARD-12 with other members of the Apaf-1/CED4 familymembers suggests a possible mechanism for its proapoptotic activity. Inthe case of both CARD-4 and Apaf-1, upstream signal-inducedself-oligomerization at the central NBS domain leads to the inducedproximity of effector molecules bound in heterotypic CARD/CARDinteractions. Apaf-1 interacts with procaspase-9 via a CARD/CARDinteraction. Oligomerization of Apaf-1 in response to mitochondrialcytochrome-c release induces proximity of bound procaspase-9 molecules,which then autoactivate, leading to downstream activation of caspase-3.This “Induced Proximity Model” of protein activation has been proposedas a general mechanism of caspase activation and of signal transductionfor signaling partners such as CARD-4/RICK, Apaf-1/Caspase-9 andFADD/Caspase-8. Based on the structural and functional data presentedhere, it is reasonable to speculate that the proapoptotic activity ofCARD-12 may occur through oligomerization of CARD-12 molecules at thecentral NBS domain in response to an upstream stress signal.CARD-12-induced cell death may then proceed in a CARD-5-dependent orpossibly caspase-1 dependent path. Similarly, CARD-12 may be involved inproinflammatory signaling by influencing caspase-1 activation ofinterleukin-1beta. TABLE 1 Summary of Human CARD-12 Sequence InformationSource of CARD-12 Predicted Predicted Predicted Sequence cDNA ProteinORF Figure cDNA SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 FIGS. sequence1A-1E genomic SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 FIGS. sequence2A-2E

TABLE 2 Summary of Domains of CARD-12 Domain Location in CARD-12 CARDabout amino acids 1-88 of SEQ ID NO: 2 NBS about amino acids 169-456 ofSEQ ID NO: 2 NACHT about amino acids 169-186 (P-Loop/Walker A Box/ MotifI); 196-220 (Walker B Box/Motif II); 229-253 (Motif III); 261-282 (MotifIV); 330-351 (Motif V); 414-430 (Motif VI) and 438-457 (Motif VII) ofSEQ ID NO: 2 Neuronal AIP about amino acids 150-1024 of SEQ ID NO: 2Homology Leucine rich about amino acids 656-686, 687-708, 711-737,repeats 738-761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930,936-962, 965-993, and 994-1021 of SEQ ID NO: 2. LRR Domain about aminoacids 656-1021 of SEQ ID NO: 2

A region, the CARD domain, of human CARD-12 protein (SEQ ID NO:2) bearssome similarity to the CARD domains of CARD-3, CARD-4, CARD-5, CARD-6,CARD-7, CARD-8, CARD-9, CARD-10, CARD-1, CARD-13, CARD-14, and CARD-15.Detailed information concerning CARD-3, CARD-4, CARD-5, CARD-6, CARD-7,CARD-8, CARD-9, CARD-10, CARD-11, CARD-13, CARD-14, and CARD-15, can befound in U.S. application Ser. No. 09/245, 281, filed Feb. 5, 1999, U.S.Pat. No. 6,369,196; U.S. application Ser. No. 09/207,359, filed Dec. 8,1998, U.S. Pat. No. 6,469,140; U.S. application Ser. No. 09/099,041,filed Jun. 17, 1998, U.S. Pat. No. 6,340,576; application Ser. No.09/019,942, filed Feb. 6, 1998, U.S. Pat. No. 6,033,855; U.S.application Ser. No. 09/428,252, filed Oct. 27, 1999, U.S. ApplicationSer. No. 60/180,021, filed Feb. 3, 2000, U.S. application Ser. No.09/573,641, filed May 17, 2000, U.S. Application Ser. No. 60/181,159filed Feb. 9, 2000, U.S. Application Ser. No. 60/168,780 filed Dec. 3,1999, U.S. application Ser. No. 09/507,533 filed Feb. 18, 2000, and U.S.application Ser. No. 09/513,904 filed Feb. 25, 2000. The entire contentof each of these applications is incorporated herein by reference.

Human CARD-12 is a member of a family of molecules (the CARD-12 family)having certain conserved structural and functional features. The term“family” when referring to the protein and nucleic acid molecules of theinvention is intended to mean two or more proteins or nucleic acidmolecules having a common structural domain and having sufficient aminoacid or nucleotide sequence identity as defined herein. Such familymembers can be naturally occurring and can be from either the same ordifferent species. For example, a family can contain a first protein ofhuman origin and a homologue of that protein of murine origin, as wellas a second, distinct protein of human origin and a murine homologue ofthat protein. Members of a family may also have common functionalcharacteristics.

Preferred CARD-12 polypeptides of the present invention include an aminoacid sequence sufficiently identical to one or more of the followingdomains: a CARD domain, an NBS domain, and a LRR domain.

As used herein, the term “sufficiently identical” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., an amino acid residue which hasa similar side chain) amino acid residues or nucleotides to a secondamino acid or nucleotide sequence such that the first and second aminoacid or nucleotide sequences have a common structural domain and/orcommon functional activity. For example, amino acid or nucleotidesequences which contain a common structural domain having about 65%identity, preferably 75% identity, more preferably 85%, 95%, or 98%identity are defined herein as sufficiently identical.

As used interchangeably herein a “CARD-12 activity”, “biologicalactivity of CARD-12” or “functional activity of CARD-12”, refers to anactivity exerted by a CARD-12 protein, polypeptide or nucleic acidmolecule on a CARD-12 responsive cell as determined in vivo, or invitro, according to standard techniques. CARD-12 may act as apro-apoptotic protein or an anti-apoptotic protein (i.e., it might actto decrease or increase apoptosis). A CARD-12 activity can be a directactivity, such as an association with or an enzymatic activity on asecond protein or an indirect activity, such as a cellular signalingactivity mediated by interaction of the CARD-12 protein with a secondprotein.

In one embodiment, a CARD-12 activity can include at least one or moreof the following activities: (i) the ability to interact with proteinsin an apoptotic signaling pathway (ii) the ability to interact with aCARD-12 ligand; or (iii) the ability to interact with an intracellulartarget protein; (iv) the ability to interact, directly or indirectlywith one or more with proteins having a CARD domain, e.g., a caspase oran AIP (e.g., AIP-1 or AIP-2); (v) the ability to modulate the activityof a caspase, e.g., caspase-9; (vi) the ability to modulate the activityof NF-κB; (vii) the ability to modulate Apaf-1; (viii) the ability tointeract directly or indirectly with a Bcl-2 family member; (ix) theability to modulate the activity of a stress activated kinase (e.g.,JNK/p38); and (x) the ability to modulate phosphorylation of CHOP (GADD153). CARD-12 nucleic acid and polypeptides as well as modulators ofactivity of expression of CARD-12 might be used to modulate an Apaf-1signaling pathway. CARD-12 may modulate the activity of a neurotrophinreceptor and thus modulate apoptosis of neuronal cells. Accordingly,CARD-12 nucleic acids and polypeptides as well as modulators of CARD-12activity or expression can be used to modulate apoptosis of neurons(e.g., for treatment of neurological disorders, particularlyneurodegenerative disorders).

Accordingly, another embodiment of the invention features isolatedCARD-12 proteins and polypeptides having a CARD-12 activity.

Various aspects of the invention are described in further detail in thefollowing subsections.

Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode CARD-12 proteins or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify CARD-12-encoding nucleic acids (e.g., CARD-12 mRNA)and fragments for use as PCR primers for the amplification or mutationof CARD-12 nucleic acid molecules. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences (preferably protein encoding sequences) that which naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated CARD-12 nucleic acid molecule can contain less than about 5 kb,4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, ora complement of any of these nucleotide sequences, can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. Using all or portion of the nucleic acid sequences ofSEQ ID NO:1, SEQ ID NO:3, as a hybridization probe, CARD-12 nucleic acidmolecules can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook et al., eds., MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid of the invention can be amplified using cDNA, mRNA orgenomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acid soamplified can be cloned into an appropriate vector and characterized byDNA sequence analysis. Furthermore, oligonucleotides corresponding toCARD-12 nucleotide sequences can be prepared by standard synthetictechniques, e.g., using an automated DNA synthesizer.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or a portionthereof. A nucleic acid molecule which is complementary to a givennucleotide sequence is one which is sufficiently complementary to thegiven nucleotide sequence that it can hybridize to the given nucleotidesequence thereby forming a stable duplex.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence encoding CARD-12, for example, afragment which can be used as a probe or primer or a fragment encoding abiologically active portion of CARD-12. The nucleotide sequencedetermined from the cloning of the human CARD-12 gene allows for thegeneration of probes and primers designed for use in identifying and/orcloning CARD-12 homologues in other cell types, e.g., from othertissues, as well as CARD-12 homologues and orthologs from other mammals.The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 75, 100,125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of thesense or anti-sense sequence of SEQ ID NO:1, SEQ ID NO:3, or of anaturally occurring mutant of one of SEQ ID NO:1 or SEQ ID NO:3.

Probes based on the CARD-12 nucleotide sequence can be used to detecttranscripts or genomic sequences encoding the same or similar proteins.The probe comprises a label group attached thereto, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying allelic variants and orthologs of the CARD-12 proteins ofthe present invention, identifying cells or tissue which mis-express aCARD-12 protein, such as by measuring a level of a CARD-12-encodingnucleic acid in a sample of cells from a subject, e.g., detectingCARD-12 mRNA levels or determining whether a genomic CARD-12 gene hasbeen mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion” ofCARD-12 can be prepared by isolating a portion of SEQ ID NO:1 or SEQ IDNO:3, which encodes a polypeptide having a CARD-12 biological activity,expressing the encoded portion of CARD-12 protein (e.g., by recombinantexpression in vitro) and assessing the activity of the encoded portionof CARD-12. For example, a nucleic acid fragment encoding a biologicallyactive portion of CARD-12 includes a CARD domain, e.g., amino acids 1-88of SEQ ID NO:2.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:3, due todegeneracy of the genetic code and thus encode the same CARD-12 proteinas that encoded by the nucleotide sequence shown in SEQ ID NO:1 or SEQID NO:3.

In addition to the CARD-12 nucleotide sequence shown in SEQ ID NO:1 andSEQ ID NO:3, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof CARD-12 may exist within a population (e.g., the human population).Such genetic polymorphism in the CARD-12 gene may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a CARD-12protein, preferably a mammalian CARD-12 protein. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of the CARD-12 gene. Any and all such nucleotide variations andresulting amino acid polymorphisms in CARD-12 that are the result ofnatural allelic variation and that do not alter the functional activityof CARD-12 are intended to be within the scope of the invention. Thus,e.g., 1%, 2%, 3%, 4%, or 5% of the amino acids in CARD-12 (e.g., 1, 2,3, 4, 5, 6, 8, 10, 15, 20, or fewer amino acids) are replaced by anotheramino acid, preferably by conservative substitution.

Moreover, nucleic acid molecules encoding CARD-12 proteins from otherspecies (CARD-12 orthologs/homologues), which have a nucleotide sequencewhich differs from that of a CARD-12 disclosed herein, are intended tobe within the scope of the invention.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 150 (300, 325, 350, 375, 400, 425, 450, 500,550, 600, 650, 700, 800, 900, 1000, 1300, 1600, 1900, 2100, 2400, 2700,3000, or 3100) nucleotides in length and hybridizes under stringentconditions to the nucleic acid molecule comprising the nucleotidesequence, preferably the coding sequence, of SEQ ID NO:1 or SEQ ID NO:3.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. An, non-limiting example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at50-65° C. (e.g., 50° C. or 60° C. or 65° C.). Preferably, the isolatednucleic acid molecule of the invention that hybridizes under stringentconditions corresponds to a naturally-occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs in ahuman cell in nature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the CARD-12sequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, thereby leading tochanges in the amino acid sequence of the encoded protein withoutaltering the functional ability of the protein. For example, one canmake nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues. A “non-essential” amino acidresidue is a residue that can be altered from the wild-type sequence ofCARD-12 protein without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the CARD-12,proteins of various species are predicted to be particularly unamenableto alteration.

For example, preferred CARD-12 proteins of the present invention containat least one CARD domain. Additionally, a CARD-12 protein also containsat least one kinase-2 domain, at least one P-loop domain, at least onenucleotide binding site domain, and at least one LRR domain. Suchconserved domains are less likely to be amenable to mutation. Otheramino acid residues, however, (e.g., those that are not conserved oronly semi-conserved among CARD-12 of various species) may not beessential for activity and thus are likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding CARD-12 proteins that contain changes in amino acidresidues that are not essential for activity. Such CARD-12 proteinsdiffer in amino acid sequence from SEQ ID NO:2, and yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule includes a nucleotide sequence encoding a protein that includesan amino acid sequence that is at least about 45% identical, 65%, 75%,85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2. Anisolated nucleic acid molecule encoding a CARD-12 protein having asequence which differs from that of SEQ ID NO:1 or SEQ ID NO:3, can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of CARD-12 (SEQ ID NO:1 or SEQID NO:3) such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. Thus, for example, 1%, 2%, 3%, 5%, or 10% of the amino acidscan be replaced by conservative substitution. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in CARD-12 is preferably replaced with another aminoacid residue from the same side chain family. Alternatively, mutationscan be introduced randomly along all or part of a CARD-12 codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for CARD-12 biological activity to identify mutants thatretain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined.

In an embodiment, a mutant CARD-12 protein can be assayed for: (1) theability to form protein:protein interactions with proteins in theapoptotic signaling pathway; (2) the ability to bind a CARD-12 ligand;or (3) the ability to bind to an intracellular target protein.

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire CARD-12 codingstrand, or to only a portion thereof, e.g., all or part of the proteincoding region (or open reading frame). An antisense nucleic acidmolecule can be antisense to a noncoding region of the coding strand ofa nucleotide sequence encoding CARD-12. The noncoding regions (“5′ and3′ untranslated regions”) are the 5′ and 3′ sequences that flank thecoding region and are not translated into amino acids. Given the codingstrand sequences encoding CARD-12 disclosed herein, antisense nucleicacids of the invention can be designed according to the rules of Watsonand Crick base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of CARD-12 mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of CARD-12 mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of CARD-12 mRNA. An antisense oligonucleotidecan be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-aino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a CARD-12protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An antisense nucleic acid molecule of the inventioncan be administered by direct injection at a tissue site. Alternatively,antisense nucleic acid molecules can be modified to target selectedcells and then administered systemically. For example, for systemicadministration, antisense molecules can be modified such that theyspecifically bind to receptors or antigens expressed on a selected cellsurface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies which bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing the vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave CARD-12 mRNA transcripts to thereby inhibittranslation of CARD-12 mRNA. A ribozyme having specificity for aCARD-12-encoding nucleic acid can be designed based upon the nucleotidesequence of a CARD-12 cDNA disclosed herein. For example, a derivativeof a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a CARD-12-encoding mRNA. See, e.g., Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, CARD-12 mRNA can be used to select a catalytic RNA havinga specific ribonuclease activity from a pool of RNA molecules. See,e.g., Bartel and Szostak (1993) Science 261:1411-1418.

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, CARD-12 gene expression can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the CARD-12 (e.g., the CARD-12 promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the CARD-12 gene in target cells. See generally, Helene (1991)Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher (1992) Bioassays 14(12):807-15.

In embodiments, the nucleic acid molecules of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4(1):5-23). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996) supra; Perry-OKeefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670-675.

PNAs of CARD-12 can be used for therapeutic and diagnostic applications.For example, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofCARD-12 can also be used, e.g., in the analysis of single base pairmutations in a gene by, e.g., PNA directed PCR clamping; as artificialrestriction enzymes when used in combination with other enzymes, e.g.,S1 nucleases (Hyrup (1996) supra; or as probes or primers for DNAsequence and hybridization (Hyrup (1996) supra; Perry-O'Keefe et al.(1996) Proc. Natl. Acad. Sci. USA 93: 14670-675).

In another embodiment, PNAs of CARD-12 can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of CARD-12 can be generated which maycombine the advantageous properties of PNA and DNA. Such chimeras allowDNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interactwith the DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996) supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996) supra and Finn et al. (1996) Nucleic Acids Research24(17):3357-63. For example, a DNA chain can be synthesized on a solidsupport using standard phosphoramidite coupling chemistry and modifiednucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used as a between the PNA and the 5′ end of DNA(Mag et al. (1989) Nucleic Acid Res. 17:5973-88). PNA monomers are thencoupled in a stepwise manner to produce a chimeric molecule with a 5′PNA segment and a 3′ DNA segment (Finn et al. (1996) Nucleic AcidsResearch 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.(1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Isolated CARD-12 Proteins and Anti-CARD-12 Antibodies.

One aspect of the invention pertains to isolated CARD-12 proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-CARD-12 antibodies. In oneembodiment, native CARD-12 proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, CARD-12 proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a CARD-12 protein or polypeptide can be synthesizedchemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theCARD-12 protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofCARD-12 protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, CARD-12 protein that is substantially free of cellularmaterial includes preparations of CARD-12 protein having less than about30%, 20%, 10%, or 5% (by dry weight) of non-CARD-12 protein (alsoreferred to herein as a “contaminating protein”). When the CARD-12protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When CARD-12 protein is produced bychemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly such preparations of CARD-12 protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors ornon-CARD-12 chemicals.

Biologically active portions of a CARD-12 protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the CARD-12 protein (e.g., the aminoacid sequence shown in SEQ ID NO:2), which include less amino acids thanthe full length CARD-12 protein, and exhibit at least one activity of aCARD-12 protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the CARD-12 protein. Abiologically active portion of a CARD-12 protein can be a polypeptidewhich is, for example, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500,600, 700, 800, 900, 1000 or more amino acids in length. Preferredbiologically active polypeptides include one or more identified CARD-12structural domains, e.g., the CARD domain (amino acids 1-88 of SEQ IDNO:2).

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativeCARD-12 protein.

CARD-12 protein has the amino acid sequence shown of SEQ ID NO:2. Otheruseful CARD-12 proteins are substantially identical to SEQ ID NO:2 andretain the functional activity of the protein of SEQ ID NO:2, yet differin amino acid sequence due to natural allelic variation or mutagenesis.

A useful CARD-12 protein is a protein which includes an amino acidsequence at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%identical to the amino acid sequence of SEQ ID NO:2, and retains thefunctional activity of the CARD-12 protein of SEQ ID NO:2.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions×100).

The determination of percent homology between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences similar or homologous to CARD-12 nucleic acidmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. When utilizing the ALIGN program forcomparing nucleic acid sequences, a gap length penalty of 12, and a gappenalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

The invention also provides CARD-12 chimeric or fusion proteins. As usedherein, a CARD-12 “chimeric protein” or “fusion protein” comprises aCARD-12 polypeptide operatively linked to a non-CARD-12 polypeptide. A“CARD-12 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to all or a portion (preferably a biologicallyactive portion) of a CARD-12, whereas a “non-CARD-12 polypeptide” refersto a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially identical to the CARD-12 protein,e.g., a protein which is different from the CARD-12 proteins and whichis derived from the same or a different organism. Within the fusionprotein, the term “operatively linked” is intended to indicate that theCARD-12 polypeptide and the non-CARD-12 polypeptide are fused in-frameto each other. The heterologous polypeptide can be fused to theN-terminus or C-terminus of the CARD-12 polypeptide.

One useful fusion protein is a GST fusion protein in which the CARD-12sequences are fused to the C-terminus of the GST sequences. Such fusionproteins can facilitate the purification of recombinant CARD-12. Inanother embodiment, the fusion protein contains a signal sequence fromanother protein. In certain host cells (e.g., mammalian host cells),expression and/or secretion of CARD-12 can be increased through use of aheterologous signal sequence. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, 1992). Other examples of eukaryotic heterologoussignal sequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal (Molecular cloning, Sambrook et al,second edition, Cold spring harbor laboratory press, 1989) and theprotein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is aCARD-12-immunoglobulin fusion protein in which all or part of CARD-12 isfused to sequences derived from a member of the immunoglobulin proteinfamily. The CARD-12-immunoglobulin fusion proteins of the invention canbe incorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a CARD-12 ligand and a CARD-12protein on the surface of a cell, to thereby suppress CARD-12-mediatedsignal transduction in vivo. The CARD-12-immunoglobulin fusion proteinscan be used to affect the bioavailability of a CARD-12 cognate ligand.Inhibition of the CARD-12 ligand/CARD-12 interaction may be usefultherapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g., promoting orinhibiting) cell survival. Moreover, the CARD-12-immunoglobulin fusionproteins of the invention can be used as immunogens to produceanti-CARD-12 antibodies in a subject, to purify CARD-12 ligands and inscreening assays to identify molecules which inhibit the interaction ofCARD-12 with a CARD-12 ligand.

Preferably, a CARD-12 chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, e.g., Current Protocols in MolecularBiology, Ausubel et al. eds., John Wiley & Sons: 1992). Moreover, manyexpression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A CARD-12-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the CARD-12 protein.

The present invention also pertains to variants of the CARD-12 proteinswhich function as either CARD-12 agonists (mimetics) or as CARD-12antagonists. Variants of the CARD-12 protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the CARD-12protein. An agonist of the CARD-12 protein can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of the CARD-12 protein. An antagonist of the CARD-12protein can inhibit one or more of the activities of the naturallyoccurring form of the CARD-12 protein by, for example, competitivelybinding to a downstream or upstream member of a cellular signalingcascade which includes the CARD-12 protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.Treatment of a subject with a variant having a subset of the biologicalactivities of the naturally occurring form of the protein can have fewerside effects in a subject relative to treatment with the naturallyoccurring form of the CARD-12 proteins.

Variants of the CARD-12 protein which function as either CARD-12agonists (mimetics) or as CARD-12 antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutantsof the CARD-12 protein for CARD-12 protein agonist or antagonistactivity. In one embodiment, a variegated library of CARD-12 variants isgenerated by combinatorial mutagenesis at the nucleic acid level and isencoded by a variegated gene library. A variegated library of CARD-12variants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential CARD-12 sequences is expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of CARD-12sequences therein. There are a variety of methods which can be used toproduce libraries of potential CARD-12 variants from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential CARD-12sequences. Methods for synthesizing degenerate oligonucleotides areknown in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura etal. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

Useful fragments of CARD-12, include fragments comprising or consistingof a domain or subdomain described herein, e.g., a kinase-2 domain or aCARD domain.

In addition, libraries of fragments of the CARD-12 protein codingsequence can be used to generate a variegated population of CARD-12fragments for screening and subsequent selection of variants of aCARD-12 protein. In one embodiment, a library of coding sequencefragments can be generated by treating a double stranded PCR fragment ofa CARD-12 coding sequence with a nuclease under conditions whereinnicking occurs only about once per molecule, denaturing the doublestranded DNA, renaturing the DNA to form double stranded DNA which caninclude sense/antisense pairs from different nicked products, removingsingle stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, an expression library can be derived whichencodes N-terminal and internal fragments of various sizes of theCARD-12 protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of CARD-12 proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify CARD-12variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

An isolated CARD-12 protein, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind CARD-12 usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length CARD-12 protein can be used or, alternatively, theinvention provides antigenic peptide fragments of CARD-12 for use asimmunogens. The antigenic peptide of CARD-12 comprises at least 8(preferably 10, 15, 20, or 30) amino acid residues of the amino acidsequence shown in SEQ ID NO:2 and encompasses an epitope of CARD-12 suchthat an antibody raised against the peptide forms a specific immunecomplex with CARD-12.

Useful antibodies include antibodies which bind to a domain or subdomainof CARD-12 described herein (e.g., a kinase-2 domain, a CARD domain, anNBS domain, a P-loop domain, or a LRR domain).

Preferred epitopes encompassed by the antigenic peptide are regions ofCARD-12 that are located on the surface of the protein, e.g.,hydrophilic regions. Other important criteria include a preference for aterminal sequence, high antigenic index (e.g., as predicted byJameson-Wolf algorithm), ease of peptide synthesis (e.g., avoidance ofprolines); and high surface probability (e.g., as predicted by the Eminialgorithm; FIG. 4).

A CARD-12 immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed CARD-12 protein or achemically synthesized CARD-12 polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic CARD-12 preparation induces a polyclonalanti-CARD-12 antibody response.

Accordingly, another aspect of the invention pertains to anti-CARD-12antibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds an antigen, such as CARD-12. A molecule whichspecifically binds to CARD-12 is a molecule which binds CARD-12, butdoes not substantially bind other molecules in a sample, e.g., abiological sample, which naturally contains CARD-12. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)2 fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind CARD-12. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of CARD-12. A monoclonal antibody composition thustypically displays a single binding affinity for a particular CARD-12protein with which it immunoreacts.

Polyclonal anti-CARD-12 antibodies can be prepared as described above byimmunizing a suitable subject with a CARD-12 immunogen. The anti-CARD-12antibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized CARD-12. If desired, the antibody moleculesdirected against CARD-12 can be isolated from the mammal (e.g., from theblood) and further purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-CARD-12 antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al.(1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96) or trioma techniques. The technology for producing variousantibodies monoclonal antibody hybridomas is well known (see generallyCurrent Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley& Sons, Inc., New York, N.Y.). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a CARD-12 immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds CARD-12.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-CARD-12 monoclonal antibody (see, e.g., Current Protocols inImmunology, supra; Galfre et al. (1977) Nature 266:55052; R. H. Kenneth,in Monoclonal Antibodies: A New Dimension In Biological Analyses, PlenumPublishing Corp., New York, N.Y. (1980); and Lerner (1981) Yale J. Biol.Med., 54:387-402). Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line, e.g., a myeloma cell line that issensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Thesemyeloma lines are available from ATCC (American Type Culture Collection,Manassas, Va.). Typically, HAT-sensitive mouse myeloma cells are fusedto mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cellsresulting from the fusion are then selected using HAT medium, whichkills unfused and unproductively fused myeloma cells (unfusedsplenocytes die after several days because they are not transformed).Hybridoma cells producing a monoclonal antibody of the invention aredetected by screening the hybridoma culture supernatants for antibodiesthat bind CARD-12, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-CARD-12 antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with CARD-12 to thereby isolateimmunoglobulin library members that bind CARD-12. Kits for generatingand screening phage display libraries are commercially available (e.g.,the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01;and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734.

Additionally, recombinant anti-CARD-12 antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNo. WO 87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-CARD-12 antibody (e.g., monoclonal antibody) can be used toisolate CARD-12 by standard techniques, such as affinity chromatographyor immunoprecipitation. An anti-CARD-12 antibody can facilitate thepurification of natural CARD-12 from cells and of recombinantly producedCARD-12 expressed in host cells. Moreover, an anti-CARD-12 antibody canbe used to detect CARD-12 protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the CARD-12 protein. Anti-CARD-12 antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, B-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response. The drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, a-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophase colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies for Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers of CytotoxicAgents in Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological and Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, and Future Prospective of TheTherapeutic Use of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation and Cytotoxic Properties of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

In addition, antibodies of the invention, either conjugated or notconjugated to a therapeutic moiety, can be administered together or incombination with a therapeutic moiety such as a cytotoxin, a therapeuticagent or a radioactive metal ion. The order of administration of theantibody and therapeutic moiety can vary. For example, in someembodiments, the antibody is administered concurrently (through the sameor different delivery devices, e.g., syringes) with the therapeuticmoiety. Alternatively, the antibody can be administered separately andprior to the therapeutic moiety. Still alternatively, the therapeuticmoiety is administered separately and prior to the antibody. In manyembodiments, these administration regimens will be continued for days,months or years.

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with a CARD-12 polypeptide, adequate to produce antibody and/or Tcell immune response to protect the animal from the diseaseshereinbefore mentioned, amongst others. Yet another aspect of theinvention relates to a method of inducing immunological response in amammal which comprises, delivering a CARD-12 polypeptide via a vectordirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect the animal from diseases.

A further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a CARD-12polypeptide of the present invention wherein the composition comprises apolypeptide or polynucleotide of CARD-12. The vaccine formulation mayfurther comprise a suitable carrier. Since a polypeptide may be brokendown in the stomach, it is preferably administered parenterally (forinstance, subcutaneous, intramuscular, intravenous, or intradermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined by routine experimentation.

Computer Readable Means

The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of mediums to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequence of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORFs)) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exist in nature or in purified form.

In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein, “computer readable media” refers to any mediumthat can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Thisskilled artisan will readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a nucleotide or aminoacid sequence of the present invention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. The skilled artisan can readily adopt anyof the presently known methods for recording information on computerreadable medium to generate manufactures comprising the nucleotide oramino acid sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a work processingtest file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order toobtain computer readable medium having recorded thereon the nucleotidesequence information of the present invention.

By providing the nucleotide or amino acid sequences of the invention incomputer readable form, the skilled artisan can routinely access thesequence information for a variety of purposes. For example, one skilledin the art can use the nucleotide or amino acid sequences of theinvention in computer readable form to compare a target sequence or atarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized thatcommercially important fragments, such as sequence fragments involved ingene expression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationformed upon the folding of the target motif. There are a variety oftarget motifs know in the art. Protein target motifs include, but arenot limited to, enzyme active sites and signal sequences. Nucleic acidtarget motifs include, but are not limited to, promoter sequences,hairpin structures and inducible expression elements (protein bindingsequences).

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knowalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware include, but is not limited to, MacPattern (EMBL), BLASTIN andBLASTX (NCBIA).

For example, software that implements the BLAST (Altschul et al. (1990)J. of Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp.Chem. 17:203-207) search algorithms on a Sybase system can be used toidentify open reading frames (ORFs) of the sequences of the inventionwhich contain homology to ORFs or proteins from other libraries. SuchORFs are protein-encoding fragments and are useful in producingcommercially important proteins such as enzymes used in variousreactions and in the production of commercially useful metabolites.

Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding CARD-12 (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of genes to which theyare operatively linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., CARD-12 proteins, mutant formsof CARD-12, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of CARD-12 in prokaryotic or eukaryotic cells, e.g.,bacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident eprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a bacterial having an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the CARD-12 expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), pGBT9 (Clontech, Palo Alto, Calif.), pGAD10 (Clontech, PaloAlto, Calif.), pYADE4 and pYGAE2 and pYPGE2 (Brunelli and Pall, (1993)Yeast 9:1299-1308), pYPGE15 (Brunelli and Pall, (1993) Yeast9:1309-1318), pACTII (Dr. S. E. Elledge, Baylor College of Medicine),and picZ (InVitrogen Corp, San Diego, Calif.). Alternatively, CARD-12can be expressed in insect cells using baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840),pCI (Promega), and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal. (supra). In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to CARD-12 mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub et al.(Reviews—Trends in Genetics, Vol. 1(1) 1986).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention or isolated nucleic acidmolecule of the invention has been introduced. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,CARD-12 protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA or an isolated nucleic acid molecule of the invention can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Insome cases vector DNA is retained by the host cell. In other cases thehost cell does not retain vector DNA and retains only an isolatednucleic acid molecule of the invention carried by the vector. In somecases, and isolated nucleic acid molecule of the invention is used totransform a cell without the use of a vector.

In order to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding CARD-12 or can be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a CARD-12protein. Accordingly, the invention further provides methods forproducing CARD-12 protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of theinvention (into which a recombinant expression vector or isolatednucleic acid molecule encoding CARD-12 has been introduced) in asuitable medium such that CARD-12 protein is produced. In anotherembodiment, the method further comprises isolating CARD-12 from themedium or the host cell.

CARD-12 nucleic acid molecules can be used in viral gene deliverysystems for gene therapy, e.g., adenoviral or retroviral gene deliverysystems.

CARD-12 nucleic acid molecules can also be used in non-viral genedelivery systems for gene therapy. Thus, another aspect of the inventionpertains to non-viral gene delivery systems, such as plasmid-based genedelivery systems. Non-viral gene delivery systems are described indetail by Huang et al. ((1999) Nonviral Vectors for Gene Therapy,Academic Press, San Diego, Calif.). Nonviral vectors have severalpotential advantages over their viral counterparts, including: reducedimmunogenicity; low acute toxicity; simplicity; and ease of large scaleproduction. Nonviral vectors can be delivered as naked DNA, bybioballistic bombardment, and in various complexes, includingliposome/DNA complexes (lipoplexes), polymer/DNA complexes (polyplexes),and liposome/polymer/DNA complexes (lipopolyplexes). Nonviral vectorsmay be administered by various routes, e.g., intravenous injection,peritoneal injection, intramuscular injection, subcutaneous injection,intratracheal injection, and aerosolization.

Naked DNA (i.e. free from association with, e.g.,transfection-facilitating proteins, viral particles, liposomalformulations, charged lipids and calcium phosphate precipitating), canbe expressed at its injection site or at a remote site. For example,naked DNA can be injected directly into skeletal muscle, liver, heartmuscle, and tumor tissue. For systemic administration, plasmid DNA mayneed to be protected from degradation by endonucleases during deliveryfrom the site of administration to the site of gene expression.

Bioballistic bombardment, also known as gene gun, allows for thepenetration of target cells in vitro, ex vivo, or in vivo. In thistechnique, DNA-coated gold particles are accelerated to a high velocityby an electric arc generated by a high voltage discharge. The method iseffective for a variety of organ types, including skin, liver, muscle,spleen, and pancreas. The gene gun transfer method is not dependent uponspecific cell surface receptors, cell cycle status, or the size of theDNA vector. Useful gene gun devices include the ACCELL® particlemediated gene gun (PowderJect Vaccines, Inc.) and the HELIOS® gene gun(Bio-Rad). These devices create a compressed shock wave of helium gas,accelerating DNA-coated gold (or tungsten) particles to high speed,whereby the particles have sufficient momentum to penetrate a targettissue.

Lipoplexes are typically made up of three components: a cationic lipid,a neutral colipid, and plasmid DNA that encodes one or more genes ofinterest. Commonly used cationic lipids include DOTMA, DMRIE, DC-chol,DOTAP, DMRIE, DDAB, DODAB/C, DOGS, DOSPA, SAINT-n, DOSPER, DPPES, DORIE,GAP-DLRIE, and DOTIM. Dioleoyl (DO) and dimyristoyl (DM) chains arethought to be especially effective for gene delivery. Cationic lipidsare typically composed of a positively charged headgroup, a hydrophobiclipid anchor, and a linker that connects the headgroup and anchor.Catioinc lipids used in lipoplexes can be divided into two broadclasses: those that use cholesterol as the lipid anchor and those thatuse diacyl chains of varying lengths and extent of saturation. Thenumber of protonatable amines on the headgroup may affect transfectionactivity, with multivalent headgroups being generally more active thanmonovalent headgroups. The linker can be made of a variety of chemicalstructures, e.g., ether, amide, carbamate, amine, urea, ester, andpeptide bonds. Neutral colipids of lipoplexes commonly include DOPE,DOPC, and cholesterol. Generally, DOPE is used as the neutral colipidwith catioinc lipids that are based on cholesterol (e.g., DC-chol,GL-67) and cholesterol is used as the neutral colipid with cationiclipids that harbor diacyl chains as the hydrophobic anchor (e.g., DOTAP,DOTIM).

Polyplexes are formed when cationic polymers are mixed with DNA.Cationic polymers used to from polyplexes are of two general types:linear polymers such as polylysine and spermine; and the branched chain,spherical, or globular polycations such as polyethyleneimine anddendrimers. Lipopolyplexes are formed by the incorporation of polylysineinto a lipoplex to form ternary complexes. DNA can be complexed with anatural biopolymer, e.g., gelatin or chitosan, functioning as a genecarrier to form nanospheres. Such biodegradable nanospheres have severaladvantages, including the coencapsulation of bioactive agents, e.g.nucleic acids and drugs, and the sustained release of the DNA.Gelatin-DNA or chitosan-DNA nanospheres are synthesized by mixing theDNA solution with an aqueous solution of gelatin or chitosan.

The effectiveness nonviral vectors may be enhanced by conjugation toligands that direct the vector either to a particular cell type or to aparticular location within a cell. Antibodies and other site-specificproteins can be attached to a vector, e.g., on the surface of the vectoror incorporated in the membrane. Following injection, these vectors bindefficiently and specifically to a target site. With respect toliposomes, ligands to a cell surface receptor can be incorporated intothe surface of a liposome by covalently modifying the ligand with alipid group and adding it during the formation of liposomes. Thefollowing classes of ligands can be incorporated into the nonviral DNAdelivery complexes of the invention in order to make them more effectivefor gene delivery: (1) peptides, e.g., peptides having a specific cellsurface receptor so that complexes will be targeted to specific cellsbearing the receptor; (2) nuclear localization signals, e.g., to promoteefficient entry of DNA into the nucleus; (3) pH-sensitive ligands, toencourage endosomal escape; (4) steric stabilizing agents, to preventdestabilization of the complexes after introduction into the biologicalmilieu. Gene chemistry approaches, e.g. peptide nucleic acids, can beused to couple ligands to DNA to improve the in vivo bioavailability andexpression of the DNA.

In plasmid-based, non-viral gene delivery systems it is often useful tolink a polypeptide (e.g., an antibody), nucleic acid molecule, or othercompound to the gene delivery plasmid such that the polypeptide, nucleicacid molecule or other compound remains associated with the plasmidfollowing intracellular delivery in a manner that does not interferewith the transcriptional activity of the plasmid. This can beaccomplished using an appropriate biotin-conjugated peptide nucleic acid(PNA) clamp. A sequence complementary to the biotin-conjugated PNA clampis inserted into the gene delivery plasmid. The biotin-conjugated PNAwill bind essentially irreversibly to the complementary sequenceinserted into the plasmid. A polypeptide, nucleic acid molecule or othercompound of interest can be conjugated to streptavidin. The streptavidinconjugate can bind to the biotin-PNA clamp bound to the plasmid. In thismanner, a polypeptide, nucleic acid molecule or other compound can bebound to a gene delivery plasmid such that the polypeptide, nucleic acidmolecule or other compound remains bound to the plasmid even within acell. Importantly, the PNA clamp-binding site in the plasmid must bechosen so as not to interfere with a needed promoter/enhancer or codingregion or otherwise disrupt the expression of the gene in the plasmid.An alternative approach employs a maleimide-conjugated PNA clamp.Polypeptides, nucleic acid molecules and other compounds containing afree thiol residue may be conjugated directly to the maleimide-PNA-DNAhybrid. As with the biotin-conjugated method, this conjugation does notdisturb the transcriptional activity of the plasmid if the PNA-bindingsite is chosen to be in a region of the plasmid not essential for geneactivity. Both of these approaches are described in detail by Zelphatiet al. ((2000) BioTechniques 28:304-315).

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichCARD-12-coding sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals in which exogenousCARD-12 sequences have been introduced into their genome or homologousrecombinant animals in which endogenous CARD-12 sequences have beenaltered. Such animals are useful for studying the function and/oractivity of CARD-12 and for identifying and/or evaluating modulators ofCARD-12 activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, an “homologous recombinant animal” is anon-human animal, preferably a mammal, more preferably a mouse, in whichan endogenous CARD-12 gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducingCARD-12-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The CARD-12cDNA sequence, e.g., that of SEQ ID NO:1 or SEQ ID NO:3 can beintroduced as a transgene into the genome of a non-human animal.Alternatively, a nonhuman homolog or ortholog of the human CARD-12 gene,such as a mouse CARD-12 gene, can be isolated based on hybridization tothe human CARD-12 cDNA and used as a transgene. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to theCARD-12 transgene to direct expression of CARD-12 protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the CARD-12 transgene in itsgenome and/or expression of CARD-12 mRNA in tissues or cells of theanimals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying a transgene encoding CARD-12 can further be bred to othertransgenic animals carrying other transgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a CARD-12 gene (e.g., a human or anon-human homolog of the CARD-12 gene, e.g., a murine CARD-12 gene) intowhich a deletion, addition or substitution has been introduced tothereby alter, e.g., functionally disrupt, the CARD-12 gene. In anembodiment, the vector is designed such that, upon homologousrecombination, the endogenous CARD-12 gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous CARD-12 gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous CARD-12 protein). In the homologousrecombination vector, the altered portion of the CARD-12 gene is flankedat its 5′ and 3′ ends by additional nucleic acid of the CARD-12 gene toallow for homologous recombination to occur between the exogenousCARD-12 gene carried by the vector and an endogenous CARD-12 gene in anembryonic stem cell. The additional flanking CARD-12 nucleic acid is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see, e.g., Thomas andCapecchi (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedCARD-12 gene has homologously recombined with the endogenous CARD-12gene are selected (see, e.g., Li et al. (1992) Cell 69:915). Theselected cells are then injected into a blastocyst of an animal (e.g., amouse) to form aggregation chimeras (see, e.g., Bradley inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach,Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA bygermline transmission of the transgene. Methods for constructinghomologous recombination vectors and homologous recombinant animals aredescribed further in Bradley (1991) Current Opinion in Bio/Technology2:823-829 and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO92/0968, and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.In brief, a cell, e.g., a somatic cell, from the transgenic animal canbe isolated and induced to exit the growth cycle and enter Go phase. Thequiescent cell can then be fused, e.g., through the use of electricalpulses, to an enucleated oocyte from an animal of the same species fromwhich the quiescent cell is isolated. The reconstructed oocyte is thencultured such that it develops to morula or blastocyte and thentransferred to pseudopregnant female foster animal. The offspring borneof this female foster animal will be a clone of the animal from whichthe cell, e.g., the somatic cell, is isolated.

Pharmaceutical Compositions

The CARD-12 nucleic acid molecules, CARD-12 proteins, and anti-CARD-12antibodies (also referred to herein as “active compounds”) of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the nucleicacid molecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide or nucleicacid of the invention. Such methods comprise formulating apharmaceutically acceptable carrier with an agent which modulatesexpression or activity of a polypeptide or nucleic acid of theinvention. Such compositions can further include additional activeagents. Thus, the invention further includes methods for preparing apharmaceutical composition by formulating a pharmaceutically acceptablecarrier with an agent which modulates expression or activity of apolypeptide or nucleic acid of the invention and one or more addtionalactive compounds.

The agent which modulates expression or activity may, for example, be asmall molecule. For example, such small molecules include peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight les thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention. Exemplary doses include milligram or microgram amountsof the small molecule per kilogram of subject or sample weight (e.g.,about 1 microgram per kilogram to about 500 milligrams per kilogram,about 100 micrograms per kilogram to about 5 milligrams per kilogram, orabout 1 microgram per kilogram to about 50 micrograms per kilogram. Itis furthermore understood that appropriate doses of a small moleculedepend upon the potency of the small molecule with respect to theexpression or activity to be modulated. Such appropriate doses may bedetermined using the assays described herein. When one or more of thesesmall molecules is to be administered to an animal (e.g., a human) inorder to modulate expression or activity of a polypeptide or nucleicacid of the invention, a physician, veterinarian, or researcher may, forexample, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular subject will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOR®EL solubilizer (BASF; Florham Park, N.J.) or phosphate buffered saline(PBS). In all cases, the composition must be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a CARD-12 protein or anti-CARD-12 antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The gene therapy vectors of the invention can be either viral ornon-viral. Examples of plasmid-based, non-viral vectors are discussed inHuang et al. (1999) Nonviral Vectors for Gene Therapy (supra). Amodified plasmid is one example of a non-viral gene delivery system.Peptides, proteins (including antibodies), and oligonucleotides may bestably conjugated to plasmid DNA by methods that do not interfere withthe transcriptional activity of the plasmid (Zelphati et al. (2000)BioTechniques 28:304-315). The attachment of proteins and/oroligonucleotides may influence the delivery and trafficking of theplasmid and thus render it a more effective pharmaceutical composition.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping, forensic biology), c) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and d) methods of treatment (e.g., therapeutic andprophylactic). A CARD-12 protein interacts with other cellular proteinsand can thus be used for (i) regulation of cellular proliferation; (ii)regulation of cellular differentiation; and (iii) regulation of cellsurvival. The isolated nucleic acid molecules of the invention can beused to express CARD-12 protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect CARD-12mRNA (e.g., in a biological sample) or a genetic lesion in a CARD-12gene, and to modulate CARD-12 activity. In addition, the CARD-12proteins can be used to screen drugs or compounds which modulate theCARD-12 activity or expression as well as to treat disorderscharacterized by insufficient or excessive production of CARD-12 proteinor production of CARD-12 protein forms which have decreased or aberrantactivity compared to CARD-12 wild type protein. In addition, theanti-CARD-12 antibodies of the invention can be used to detect andisolate CARD-12 proteins and modulate CARD-12 activity.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to CARD-12 proteins or biologically active portions thereofor have a stimulatory or inhibitory effect on, for example, CARD-12expression or CARD-12 activity. An example of a biologically activeportion of human CARD-12 is amino acids 139-227 encoding a CARD domain.

Among the screening assays provided by the invention are screening toidentify molecules that prevent the dimerization of CARD-12, screeningto identify molecules which block the binding of a CARD containingpolypeptide to CARD-12, and screening to identify a competitiveinhibitor of the binding of a nucleotide to the nucleotide binding siteof CARD-12. Screening assays, e.g., dimerization assays, can employfull-length CARD-12 or a portion of CARD-12, e.g, the CARD domain, thenucleotide binding site domain, or the NAIP homology domain.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a CARD-12proteins or polypeptides or biologically active portions thereof. Thetest compounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145). Examples of methods for the synthesis of molecular librariescan be found in the art, for example in: DeWitt et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad.Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Choet al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int.Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).

Determining the ability of the test compound to modulate the activity ofCARD-12 or a biologically active portion thereof can be accomplished,for example, by determining the ability of the CARD-12 protein to bindto or interact with a CARD-12 target molecule. As used herein, a “targetmolecule” is a molecule with which a CARD-12 protein binds or interactsin nature, for example, a molecule associated with the internal surfaceof a cell membrane or a cytoplasmic molecule. A CARD-12 target moleculecan be a non-CARD-12 molecule or a CARD-12 protein or polypeptide of thepresent invention. In one embodiment, a CARD-12 target molecule is acomponent of an apoptotic signal transduction pathway. The target, forexample, can be a second intracellular protein which has catalyticactivity or a protein which facilitates the association of downstreamsignaling molecules with CARD-12.

Determining the ability of the test compound to modulate the activity ofCARD-12 or a biologically active portion thereof can be accomplished,for example, by determining the ability of the CARD-12 protein to bindto or interact with any of the specific proteins listed in the previousparagraph as CARD-12 target molecules. In another embodiment, CARD-12target molecules include all proteins that bind to a CARD-12 protein ora fragment thereof in a two-hybrid system binding assay which can beused without undue experimentation to isolate such proteins from cDNA orgenomic two-hybrid system libraries. The binding assays described inthis section can be cell-based or cell free (described subsequently).

Determining the ability of the CARD-12 protein to bind to or interactwith a CARD-12 target molecule can be accomplished by one of the methodsdescribed above for determining direct binding. In an embodiment,determining the ability of the CARD-12 protein to bind to or interactwith a CARD-12 target molecule can be accomplished by determining theactivity of the target molecule. For example, the activity of the targetmolecule can be determined by detecting induction of a cellular secondmessenger of the target (e.g., intracellular Ca2+, diacylglycerol, IP3,etc.), detecting catalytic/enzymatic activity of the target on anappropriate substrate, detecting the induction of a reporter gene (e.g.,a CARD-12-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g. luciferase), or detecting acellular response, for example, cell survival, cellular differentiation,or cell proliferation. In addition, and in another embodiment, genesinduced by CARD-12 expression can be identified by expressing CARD-12 ina cell line and conducting a transcriptional profiling experimentwherein the mRNA expression patterns of the cell line transformed withan empty expression vector and the cell line transformed with a CARD-12expression vector are compared. The promoters of genes induced byCARD-12 expression can be operatively linked to reporter genes suitablefor screening such as luciferase, secreted alkaline phosphatase, orbeta-galactosidase and the resulting constructs could be introduced intoappropriate expression vectors. A recombinant cell line containingCARD-12 and transfected with an expression vector containing a CARD-12responsive promoter operatively linked to a reporter gene can be used toidentify test compounds that modulate CARD-12 activity by assaying theexpression of the reporter gene in response to contacting therecombinant cell line with test compounds. CARD-12 agonists can beidentified as increasing the expression of the reporter gene and CARD-12antagonists can be identified as decreasing the expression of thereporter gene.

In another embodiment of the invention, the ability of a test compoundto modulate the activity of CARD-12, or biologically active portionsthereof can be determined by assaying the ability of the test compoundto modulate CARD-12-dependent pathways or processes where the CARD-12target proteins that mediate the CARD-12 effect are known or unknown.Potential CARD-12-dependent pathways or processes include, but are notlimited to, the modulation of cellular signal transduction pathways andtheir related second messenger molecules (e.g., intracellular Ca2+,diacylglycerol, IP3, cAMP etc.), cellular enzymatic activities, cellularresponses (e.g., cell survival, cellular differentiation, or cellproliferation), or the induction or repression of cellular orheterologous mRNAs or proteins. CARD-12-dependent pathways or processescould be assayed by standard cell-based or cell free assays appropriatefor the specific pathway or process under study.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a CARD-12 protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the CARD-12 protein or biologicallyactive portion thereof. Binding of the test compound to the CARD-12protein can be determined either directly or indirectly as describedabove. In one embodiment, a competitive binding assay includescontacting the CARD-12 protein or biologically active portion thereofwith a compound known to bind CARD-12 to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a CARD-12 protein, whereindetermining the ability of the test compound to interact with a CARD-12protein comprises determining the ability of the test compound topreferentially bind to CARD-12 or biologically active portion thereof ascompared to the known binding compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting CARD-12 protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the CARD-12protein or biologically active portion thereof. Determining the abilityof the test compound to modulate the activity of CARD-12 can beaccomplished, for example, by determining the ability of the CARD-12protein to bind to a CARD-12 target molecule by one of the methodsdescribed above for determining direct binding. In an alternativeembodiment, determining the ability of the test compound to modulate theactivity of CARD-12 can be accomplished by determining the ability ofthe CARD-12 protein to further modulate a CARD-12 target molecule. Forexample, the catalytic/enzymatic activity of the target molecule on anappropriate substrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theCARD-12 protein or biologically active portion thereof with a knowncompound which binds CARD-12 to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a CARD-12 protein, wherein determiningthe ability of the test compound to interact with a CARD-12 proteincomprises determining the ability of the CARD-12 protein topreferentially bind to or modulate the activity of a CARD-12 targetmolecule. The cell-free assays of the present invention are amenable touse of either the soluble form or a membrane-associated form of CARD-12.A membrane-associated form of CARD-12 refers to CARD-12 that interactswith a membrane-bound target molecule. In the case of cell-free assayscomprising the membrane-associated form of CARD-12, it may be desirableto utilize a solubilizing agent such that the membrane-associated formof CARD-12 is maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either CARD-12 or itstarget molecule to facilitate separation of complexed from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of a test compound to CARD-12, orinteraction of CARD-12 with a target molecule in the presence andabsence of a candidate compound, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotitre plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-5-transferase/CARD-12 fusion proteins orglutathione-5-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or CARD-12 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofCARD-12 binding or activity determined using standard techniques. In analternative embodiment, MYC or HA epitope tag CARD-12 fusion proteins orMYC or HA epitope tag target fusion proteins can be adsorbed ontoanti-MYC or anti-HA antibody coated microbeads or onto anti-MYC oranti-HA antibody coated microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or CARD-12 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofCARD-12 binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, CARD-12 or itstarget molecule can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated CARD-12 target molecules can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques well known in the art(e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with CARD-12 or targetmolecules but which do not interfere with binding of the protein to itstarget molecule can be derivatized to the wells of the plate, andunbound target or protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes and epitope tag immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the CARD-12 or target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with theCARD-12 or a target molecule.

In another embodiment, modulators of CARD-12 expression are identifiedin a method in which a cell is contacted with a candidate compound andthe expression of the CARD-12 promoter, mRNA or protein in the cell isdetermined. The level of expression of CARD-12 mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of CARD-12 mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof CARD-12 expression based on this comparison. For example, whenexpression of CARD-12 mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofCARD-12 mRNA or protein expression. Alternatively, when expression ofCARD-12 mRNA or protein is less (statistically significantly less) inthe presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of CARD-12 mRNA orprotein expression. The level of CARD-12 mRNA or protein expression inthe cells can be determined by methods described herein for detectingCARD-12 mRNA or protein. The activity of the CARD-12 promoter can beassayed by linking the CARD-12 promoter to a reporter gene such asluciferase, secreted alkaline phosphatase, or beta-galactosidase andintroducing the resulting construct into an appropriate vector,transfecting a host cell line, and measuring the activity of thereporter gene in response to test compounds.

In yet another aspect of the invention, the CARD-12 proteins can be usedas “bait proteins” in a two-hybrid assay or three hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with CARD-12 (“CARD-12-bindingproteins” or “CARD-12-bp”) and modulate CARD-12 activity. SuchCARD-12-binding proteins are also likely to be involved in thepropagation of signals by the CARD-12 proteins as, for example, upstreamor downstream elements of the CARD-12 pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for CARD-12 is fusedto a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a CARD-12-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with CARD-12.

In an embodiment of the invention, the ability of a test compound tomodulate the activity of CARD-12, or a biologically active portionthereof can be determined by assaying the ability of the test compoundto block the binding of CARD-12 to its target proteins in a yeast ormammalian two-hybrid system assay. This assay could be automated forhigh throughput drug screening purposes. In another embodiment of theinvention, CARD-12 and a target protein could be configured in thereverse two-hybrid system (Vidal et al. (1996) Proc. Natl. Acad. Sci.USA 93:10321-6 and Vidal et al. (1996) Proc. Natl. Acad. Sci. USA93:10315-20) designed specifically for efficient drug screening. In thereverse two-hybrid system, inhibition of a CARD-12 physical interactionwith a target protein would result in induction of a reporter gene incontrast to the normal two-hybrid system where inhibition of CARD-12physical interaction with a target protein would lead to reporter generepression. The reverse two-hybrid system is preferred for drugscreening because reporter gene induction is more easily assayed thanreport gene repression.

Alternative embodiments of the invention are proteins found tophysically interact with proteins that bind to CARD-12. CARD-12interactors could be configured into two-hybrid system baits and used intwo-hybrid screens to identify additional members of the CARD-12pathway. The interactors of CARD-12 interactors identified in this waycould be useful targets for therapeutic intervention in CARD-12 relateddiseases and pathologies and an assay of their enzymatic or bindingactivity could be useful for the identification of test compounds thatmodulate CARD-12 activity.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. Accordingly, CARD-12 nucleic acid molecules described hereinor fragments thereof, can be used to map the location of CARD-12 geneson a chromosome. The mapping of the CARD-12 sequences to chromosomes isan important first step in correlating these sequences with genesassociated with disease.

Briefly, CARD-12 genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the CARD-12 sequences.Computer analysis of CARD-12 sequences can be used to rapidly selectprimers that do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers can then be usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the CARD-12 sequences will yield an amplified fragment. Somatic cellhybrids are prepared by fusing somatic cells from different mammals(e.g., human and mouse cells). As hybrids of human and mouse cells growand divide, they gradually lose human chromosomes in random order, butretain the mouse chromosomes. By using media in which mouse cells cannotgrow, because they lack a particular enzyme, but human cells can, theone human chromosome that contains the gene encoding the needed enzyme,will be retained. By using various media, panels of hybrid cell linescan be established. Each cell line in a panel contains either a singlehuman chromosome or a small number of human chromosomes, and a full setof mouse chromosomes, allowing easy mapping of individual genes tospecific human chromosomes. (D'Eustachio et al. (1983) Science220:919-924). Somatic cell hybrids containing only fragments of humanchromosomes can also be produced using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the CARD-12sequences to design oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes. Othermapping strategies which can similarly be used to map a CARD-12 sequenceto its chromosome include in situ hybridization (described in Fan et al.(1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening withlabeled flow-sorted chromosomes, and pre-selection by hybridization tochromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., (Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York, 1988)).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature,325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the CARD-12 gene can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

A CARD-12 polypeptide and fragments and sequences thereof and antibodiesspecific thereto can be used to map the location of the gene encodingthe polypeptide on a chromosome. This mapping can be carried out byspecifically detecting the presence of the polypeptide in members of apanel of somatic cell hybrids between cells of a first species of animalfrom which the protein originates and cells from a second species ofanimal and then determining which somatic cell hybrid(s) expresses thepolypeptide and noting the chromosome(s) from the first species ofanimal that it contains. For examples of this technique, see Pajunen etal. (1988) Cytogenet. Cell Genet. 47:37-41 and Van Keuren et al. (1986)Hum. Genet. 74:34-40. Alternatively, the presence of the CARD-12polypeptide in the somatic cell hybrids can be determined by assaying anactivity or property of the polypeptide, for example, enzymaticactivity, as described in Bordelon-Riser et al. (1979) Somatic CellGenetics 5:597-613 and Owerbach et al. (1978) Proc. Natl. Acad. Sci. USA75:5640-5644.

Tissue Typing

The CARD-12 sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the CARD-12 sequences described herein can be used toprepare two PCR primers from the 5′ and 3′ ends of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The CARD-12 sequences of the invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Each ofthe sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1 can comfortablyprovide positive individual identification with a panel of perhaps 10 to1,000 primers which each yield a noncoding amplified sequence of 100bases. If predicted coding sequences, such as those in SEQ ID NO:3 areused, a more appropriate number of primers for positive individualidentification would be 500-2,000.

If a panel of reagents from CARD-12 sequences described herein is usedto generate a unique identification database for an individual, thosesame reagents can later be used to identify tissue from that individual.Using the unique identification database, positive identification of theindividual, living or dead, can be made from extremely small tissuesamples.

Use of Partial Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include theCARD-12 sequences or portions thereof, e.g., fragments derived from thenoncoding regions of SEQ ID NO:1 which have a length of at least 20 or30 bases.

The sequences described herein can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes which can beused in, for example, an in situ hybridization technique, to identify aspecific tissue, e.g., brain tissue. This can be very useful in caseswhere a forensic pathologist is presented with a tissue of unknownorigin. Panels of such CARD-12 probes can be used to identify tissue byspecies and/or by organ type.

In a similar fashion, these reagents, e.g., CARD-12 primers or probescan be used to screen tissue culture for contamination (i.e., screen forthe presence of a mixture of different types of cells in a culture).

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trials are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningCARD-12 protein and/or nucleic acid expression as well as CARD-12activity, in the context of a biological sample (e.g., blood, serum,cells, tissue) to thereby determine whether an individual is afflictedwith a disease or disorder, or is at risk of developing a disorder,associated with aberrant CARD-12 expression or activity. The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing a disorder associatedwith CARD-12 protein, nucleic acid expression or activity. For example,mutations in a CARD-12 gene can be assayed in a biological sample. Suchassays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with CARD-12 protein, nucleic acidexpression or activity.

Another aspect of the invention provides methods for determining CARD-12protein, nucleic acid expression or CARD-12 activity in an individual tothereby select appropriate therapeutic or prophylactic agents for thatindividual (referred to herein as “pharmacogenomics”). Pharmacogenomicsallows for the selection of agents (e.g., drugs) for therapeutic orprophylactic treatment of an individual based on the genotype of theindividual (e.g., the genotype of the individual examined to determinethe ability of the individual to respond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds) on the expression or activityof CARD-12 in clinical trials.

These and other agents are described in further detail in the followingsections.

Diagnostic Assays

An exemplary method for detecting the presence or absence of CARD-12 ina biological sample involves obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting CARD-12 protein or nucleic acid (e.g., mRNA,genomic DNA) that encodes CARD-12 protein such that the presence ofCARD-12 is detected in the biological sample. An agent for detectingCARD-12 mRNA or genomic DNA is a labeled nucleic acid probe capable ofhybridizing to CARD-12 mRNA or genomic DNA. The nucleic acid probe canbe, for example, a full-length CARD-12 nucleic acid, such as the nucleicacid of SEQ ID NO:1 or 3, or a portion thereof, such as anoligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to mRNA or genomic DNA. Other suitable probes for use in thediagnostic assays of the invention are described herein.

An agent for detecting CARD-12 protein can be an antibody capable ofbinding to CARD-12 protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect CARD-12 mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of CARD-12 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of CARD-12 protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of CARD-12 genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of CARD-12 protein include introducing into a subject alabeled anti-CARD-12 antibody. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A biological sample is a peripheral blood leukocyte sampleisolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting CARD-12 protein, mRNA, orgenomic DNA, such that the presence of CARD-12 protein, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofCARD-12 protein, mRNA or genomic DNA in the control sample with thepresence of CARD-12 protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence ofCARD-12 in a biological sample (a test sample). Such kits can be used todetermine if a subject is suffering from or is at increased risk ofdeveloping a disorder associated with aberrant expression of CARD-12(e.g., an immunological disorder). For example, the kit can comprise alabeled compound or agent capable of detecting CARD-12 protein or mRNAin a biological sample and means for determining the amount of CARD-12in the sample (e.g., an anti-CARD-12 antibody or an oligonucleotideprobe which binds to DNA encoding CARD-12, e.g., SEQ ID NO:1 or SEQ IDNO:3). Kits may also include instruction for observing that the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of CARD-12 if the amount of CARD-12protein or mRNA is above or below a normal level.

For antibody-based kits, the kit may comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to CARD-12protein; and, optionally, (2) a second, different antibody which bindsto CARD-12 protein or the first antibody and is conjugated to adetectable agent. For oligonucleotide-based kits, the kit may comprise,for example: (1) a oligonucleotide, e.g., a detectably labelledoligonucleotide, which hybridizes to a CARD-12 nucleic acid sequence or(2) a pair of primers useful for amplifying a CARD-12 nucleic acidmolecule.

The kit may also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit may also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit may also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of CARD-12.

Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant CARD-12expression or activity. For example, the assays described herein, suchas the preceding diagnostic assays or the following assays, can beutilized to identify a subject having or at risk of developing adisorder associated with CARD-12 protein, nucleic acid expression oractivity. Alternatively, the prognostic assays can be utilized toidentify a subject having or at risk for developing such a disease ordisorder. Thus, the present invention provides a method in which a testsample is obtained from a subject and CARD-12 protein or nucleic acid(e.g., mRNA, genomic DNA) is detected, wherein the presence of CARD-12protein or nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant CARD-12expression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue. Furthermore, the prognostic assays described herein can be usedto determine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant CARD-12 expression or activity. For example,such methods can be used to determine whether a subject can beeffectively treated with a specific agent or class of agents (e.g.,agents of a type which decrease CARD-12 activity). Thus, the presentinvention provides methods for determining whether a subject can beeffectively treated with an agent for a disorder associated withaberrant CARD-12 expression or activity in which a test sample isobtained and CARD-12 protein or nucleic acid is detected (e.g., whereinthe presence of CARD-12 protein or nucleic acid is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant CARD-12 expression or activity).

The methods of the invention can also be used to detect genetic lesionsor mutations in a CARD-12 gene, thereby determining if a subject withthe lesioned gene is at risk for a disorder characterized by aberrantcell proliferation and/or differentiation. In preferred embodiments, themethods include detecting, in a sample of cells from the subject, thepresence or absence of a genetic lesion characterized by at least one ofan alteration affecting the integrity of a gene encoding aCARD-12-protein, or the mis-expression of the CARD-12 gene. For example,such genetic lesions can be detected by ascertaining the existence of atleast one of 1) a deletion of one or more nucleotides from a CARD-12gene; 2) an addition of one or more nucleotides to a CARD-12 gene; 3) asubstitution of one or more nucleotides of a CARD-12 gene; 4) achromosomal rearrangement of a CARD-12 gene; 5) an alteration in thelevel of a messenger RNA transcript of a CARD-12 gene; 6) aberrantmodification of a CARD-12 gene, such as of the methylation pattern ofthe genomic DNA; 7) the presence of a non-wild type splicing pattern ofa messenger RNA transcript of a CARD-12 gene (e.g., caused by a mutationin a splice donor or splice acceptor site); 8) a non-wild type level ofa CARD-12-protein; 9) allelic loss of a CARD-12 gene; and 10)inappropriate post-translational modification of a CARD-12-protein. Asdescribed herein, there are a large number of assay techniques known inthe art which can be used for detecting lesions in a CARD-12 gene. Abiological sample is a peripheral blood leukocyte sample isolated byconventional means from a subject.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the CARD-12 gene(see, e.g., Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a CARD-12 gene under conditionssuch that hybridization and amplification of the CARD-12-gene (ifpresent) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a CARD-12 gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, e.g., U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In other embodiments, genetic mutations in CARD-12 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al.(1996) Nature Medicine 2:753-759). For example, genetic mutations inCARD-12 can be identified in two-dimensional arrays containinglight-generated DNA probes as described in Cronin et al. supra. Briefly,a first hybridization array of probes can be used to scan through longstretches of DNA in a sample and control to identify base changesbetween the sequences by making linear arrays of sequential overlappingprobes. This step allows the identification of point mutations. Thisstep is followed by a second hybridization array that allows thecharacterization of specific mutations by using smaller, specializedprobe arrays complementary to all variants or mutations detected. Eachmutation array is composed of parallel probe sets, one complementary tothe wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the CARD-12 gene anddetect mutations by comparing the sequence of the sample CARD-12 withthe corresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the CARD-12 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type CARD-12 sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, e.g., Cottonet al (1988) Proc. Natl. Acad Sci USA 85:4397; Saleeba et al (1992)Methods Enzymol. 217:286-295. In an embodiment, the control DNA or RNAcan be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in CARD-12 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a CARD-12sequence, e.g., a wild-type CARD-12 sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, e.g., U.S.Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in CARD-12 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control CARD-12nucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In an embodiment,the subject method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. SciUSA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a CARD-12 gene.

Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which CARD-12 is expressed may be utilized in theprognostic assays described herein.

Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect onCARD-12 activity (e.g., CARD-12 gene expression) as identified by ascreening assay described herein can be administered to individuals totreat (prophylactically or therapeutically) disorders (e.g., animmunological disorder) associated with aberrant CARD-12 activity. Inconjunction with such treatment, the pharmacogenomics (i.e., the studyof the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual's genotype. Suchpharmacogenomics can further be used to determine appropriate dosagesand therapeutic regimens. Accordingly, the activity of CARD-12 protein,expression of CARD-12 nucleic acid, or mutation content of CARD-12 genesin an individual can be determined to thereby select appropriateagent(s) for therapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM exhibit no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so-called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of CARD-12 protein, expression of CARD-12 nucleicacid, or mutation content of CARD-12 genes in an individual can bedetermined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a CARD-12 modulator, such as a modulator identified by one of theexemplary screening assays described herein.

Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of CARD-12 (e.g., the ability to modulateaberrant cell proliferation and/or differentiation) can be applied notonly in basic drug screening, but also in clinical trials. For example,the effectiveness of an agent determined by a screening assay asdescribed herein to increase CARD-12 gene expression, protein levels, orupregulate CARD-12 activity, can be monitored in clinical trails ofsubjects exhibiting decreased CARD-12 gene expression, protein levels,or downregulated CARD-12 activity. Alternatively, the effectiveness ofan agent determined by a screening assay to decrease CARD-12 geneexpression, protein levels, or downregulated CARD-12 activity, can bemonitored in clinical trials of subjects exhibiting increased CARD-12gene expression, protein levels, or upregulated CARD-12 activity. Insuch clinical trials, the expression or activity of CARD-12 and,preferably, other genes that have been implicated in, for example, acellular proliferation disorder can be used as a “read out” or markersof the immune responsiveness of a particular cell.

For example, and not by way of limitation, genes, including CARD-12,that are modulated in cells by treatment with an agent (e.g., compound,drug or small molecule) which modulates CARD-12 activity (e.g.,identified in a screening assay as described herein) can be identified.Thus, to study the effect of agents on cellular proliferation disorders,for example, in a clinical trial, cells can be isolated and RNA preparedand analyzed for the levels of expression of CARD-12 and other genesimplicated in the disorder. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of CARD-12 or other genes. In this way,the gene expression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the agent.

In an embodiment, the present invention provides a method for monitoringthe effectiveness of treatment of a subject with an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate identified by the screeningassays described herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a CARD-12 protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the CARD-12 protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the CARD-12 protein, mRNA, or genomic DNAin the pre-administration sample with the CARD-12 protein, mRNA, orgenomic DNA in the post administration sample or samples; and (vi)altering the administration of the agent to the subject accordingly. Forexample, increased administration of the agent may be desirable toincrease the expression or activity of CARD-12 to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of CARD-12 to lower levels thandetected, i.e., to decrease the effectiveness of the agent.

Transcriptional Profiling

The CARD-12 nucleic acid molecules described herein, including smalloligonucleotides, can be used in transcriptionally profiling. Forexample, these nucleic acids can be used to examine the expression ofCARD-12 in normal tissue or cells and in tissue or cells subject to adisease state, e.g., tissue or cells derived from a patient having adisease of interest or cultured cells which model or reflect a diseasestate of interest, e.g., cells of a cultured tumor cell line. Bymeasuring expression of CARD-12, together or individually, a profile ofexpression in normal and disease states can be developed. This profilecan be used diagnostically and to examine the effectiveness of atherapeutic regime.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant CARD-12 expression oractivity, examples of which are provided herein.

Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant CARD-12expression or activity, by administering to the subject an agent whichmodulates CARD-12 expression or at least one CARD-12 activity. Subjectsat risk for a disease which is caused or contributed to by aberrantCARD-12 expression or activity can be identified by, for example, any ora combination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the CARD-12 aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of CARD-12 aberrancy, forexample, a CARD-12 agonist or CARD-12 antagonist agent can be used fortreating the subject. The appropriate agent can be determined based onscreening assays described herein.

Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingCARD-12 expression or activity for therapeutic purposes. The modulatorymethod of the invention involves contacting a cell with an agent thatmodulates one or more of the activities of CARD-12 protein activityassociated with the cell. An agent that modulates CARD-12 proteinactivity can be an agent as described herein, such as a nucleic acid ora protein, a naturally-occurring cognate ligand of a CARD-12 protein, apeptide, a CARD-12 peptidomimetic, or other small molecule. In oneembodiment, the agent stimulates one or more of the biologicalactivities of CARD-12 protein. Examples of such stimulatory agentsinclude active CARD-12 protein and a nucleic acid molecule encodingCARD-12 that has been introduced into the cell. In another embodiment,the agent inhibits one or more of the biological activities of CARD-12protein. Examples of such inhibitory agents include antisense CARD-12nucleic acid molecules and anti-CARD-12 antibodies. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a CARD-12 protein or nucleic acidmolecule or a disorder related to CARD-12 expression or activity. In oneembodiment, the method involves administering an agent (e.g., an agentidentified by a screening assay described herein), or combination ofagents that modulates (e.g., upregulates or downregulates) CARD-12expression or activity. In another embodiment, the method involvesadministering a CARD-12 protein or nucleic acid molecule as therapy tocompensate for reduced or aberrant CARD-12 expression or activity.

Stimulation of CARD-12 activity is desirable in situations in whichCARD-12 is abnormally downregulated and/or in which increased CARD-12activity is likely to have a beneficial effect. Conversely, inhibitionof CARD-12 activity is desirable in situations in which CARD-12 isabnormally upregulated, e.g., in myocardial infarction, and/or in whichdecreased CARD-12 activity is likely to have a beneficial effect.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:2; b) a polypeptide comprising an amino acid sequence at least 90% identical to the CARD domain (amino acid residues 1-88 of SEQ ID NO:2) of CARD-12; c) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 161-323 of SEQ ID NO:2 of CARD-12; d) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 762-965 of SEQ ID NO:2 of CARD-12; e) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 169-456 of SEQ ID NO:2) of CARD-12; f) a polypeptide with a length of at least 25 amino acids, comprising at least one NACHT NTPase domain (amino acid residues 169-186, 196-220, 229-253, 261-282, 330-351, 414-430, or 438-457 of SEQ ID NO:2) of CARD-12; g) a polypeptide with a length of at least 50 amino acids, comprising at least one leucine-rich repeat domain (amino acid residues 656-686, 687-708, 711-737, 738-761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930, 936-962, 965-993, or 994-1021 of SEQ ID NO:2) of CARD-12; and h) a polypeptide with a length of at least 25 amino acids, comprising the P-loop (amino acid residues 169-179 of SEQ ID NO:2) of CARD-12.
 2. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2 wherein up to 10 amino acids are replaced by conservative substitutions.
 3. A fusion protein comprising the polypeptide of claim 1 and a non-CARD-12 polypeptide.
 4. The fusion protein of claim 3, wherein the non-CARD-12 polypeptide is glutathione S-transferase.
 5. A method for detecting the presence of a polypeptide of claim 1 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of claim 1; and b) determining whether the compound binds to the polypeptide in the sample.
 6. The method of claim 5, wherein the compound that binds to the polypeptide is an antibody.
 7. A method for identifying a compound which binds to CARD-12, the method comprising the steps of: a) contacting a polypeptide, or a cell from the group consisting of: i) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:2; ii) a polypeptide comprising an amino acid sequence at least 90% identical to the CARD domain (amino acid residues 1-88 of SEQ ID NO:2) of CARD-12; iii) a polypeptide comprising an amino acid sequence at least 90% identical to an NBS domain (amino acid residues 161-323 of SEQ ID NO:2) of CARD-12; iv) a polypeptide comprising an amino acid sequence at least 90% identical to a LRR domain (amino acid residues 762-965 of SEQ ID NO:2) of CARD-12; v) a polypeptide comprising an amino acid sequence of a fragment of at least 200 amino acids of SEQ ID NO:2, wherein the fragment has at least one binding activity of CARD-12; and vi) a cell expressing the polypeptide of i), ii), iii), iv) or v), with a test compound; and b) determining whether the polypeptide binds to the test compound.
 8. The method of claim 7, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; b) detection of binding using a competition binding assay; c) detection of binding using an assay for CARD-12-mediated signal transduction; d) detection of an effect on the binding of the polypeptide to a CARD domain; e) detection of an effect on the binding of the polypeptide to another protein; and f) detection of an effect on the binding of the polypeptide to a nucleotide.
 9. The method of claim 7, wherein the fragment includes a polypeptide selected from the group consisting of: a) a polypeptide comprising an NBS domain (amino acid residues 169-456 of SEQ ID NO:2) of CARD-12; b) a polypeptide comprising at least one NACHT NTPase domain (amino acid residues 169-186, 196-220, 229-253, 261-282, 330-351, 414-430, or 438-457 of SEQ ID NO:2) of CARD-12; c) a polypeptide comprising a LRR domain (amino acid residues 656-1021 of SEQ ID NO:2) of CARD-12; and d) a polypeptide comprising at least one leucine-rich repeat domain (amino acid residues 656-686, 687-708, 711-737, 738-761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930, 936-962, 965-993, or 994-1021) of CARD-12.
 10. The method of claim 7, wherein the polypeptide is operatively linked to a non-CARD-12 polypeptide.
 11. The method of claim 7, wherein the test compound is contacted with a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a cell expressing a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
 12. The method of claim 8, wherein the detection of an effect on the binding of the polypeptide to a CARD domain comprises detection of an effect on the binding of CARD-12 to a CARD-5 polypeptide, wherein the CARD-5 polypeptide comprises the CARD domain of CARD-5.
 13. The method of claim 8, wherein the detection of an effect on the binding of the polypeptide to a CARD domain comprises detection of an effect on the binding of CARD-12 to a CARD-12 polypeptide, wherein the CARD-12 polypeptide comprises the CARD domain of CARD-12.
 14. The method of claim 8, wherein the detection of an effect on the binding of the polypeptide to another protein comprises detection of an effect on the binding of CARD-12 to caspase-1.
 15. A method for identifying a candidate compound for treating an apoptotic disorder or an inflammatory disorder, the method comprising: a) measuring the binding of a first polypeptide or cell selected from the group consisting of: i) a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:2; ii) a polypeptide comprising an amino acid sequence at least 90% identical to the CARD domain (amino acid residues 1-88 of SEQ ID NO:2) of CARD-12; iii) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 161-323 of SEQ ID NO:2 of CARD-12; iv) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 762-965 of SEQ ID NO:2 of CARD-12; v) a polypeptide comprising an amino acid sequence at least 90% identical to amino acid residues 169-456 of SEQ ID NO:2) of CARD-12; vi) a polypeptide with a length of at least 25 amino acids, comprising at least one NACHT NTPase domain (amino acid residues 169-186, 196-220, 229-253, 261-282,330-351, 414-430, or 438-457 of SEQ ID NO:2) of CARD-12; vii) a polypeptide with a length of at least 50 amino acids, comprising at least one leucine-rich repeat domain (amino acid residues 656-686, 687-708, 711-737, 738-761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930, 936-962, 965-993, or 994-1021 of SEQ ID NO:2) of CARD-12; and viii) a cell expressing the polypeptide of i), ii), iii), iv), v) vi) or vii), to a second molecule selected from the group consisting of: a polypeptide comprising a CARD domain, caspase-1 and a nucleotide, in the presence of a test compound; and b) comparing the binding of the first polypeptide or cell to the second molecule measured in step (a) to the binding of the first polypeptide or cell to the second molecule in the absence of the test compound, wherein altered binding of the first polypeptide or cell to the second molecule in the presence of the test compound compared the binding in the absence of the test compound indicates that the test compound is candidate compound treating a disorder.
 16. The method of claim 15, wherein the apoptotic disorder is a cancer, an autoimmune disorder, a viral infection, Alzheimer's disease, Parkinson's disease or aplastic anemia.
 17. The method of claim 15, wherein the inflammatory disorder is arthritis, Crohn's disease, psoriasis, allergy or asthma.
 18. The method of claim 15, wherein the test compound is contacted with a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a cell expressing a polypeptide comprising the amino acid sequence of SEQ ID NO:2. 